悬浮芯片联合高效液相色谱检测乳腺癌血清标志物的研究
|
摘要
背景
乳腺癌是全世界女性最常见的恶性肿瘤之一,严重威胁女性的身心健康。全世界乳腺癌的发病率一直呈稳步上升的趋势,20世纪末的统计资料表明世界每年约有115万人诊断为乳腺癌,有41万人死于该病,发病年龄也有年轻化趋势,是导致女性肿瘤患者死亡的第一大原因。然而,乳腺癌的病因尚不十分清楚,其发病机制也非常复杂,影响发病的因素很多。近年来,随着肿瘤分子病理学和分子生物学研究的深入,人们认识到肿瘤的发生和发展是一个多阶段、多基因改变参与的渐进过程。多个癌基因的异常激活和编码蛋白的过度表达以及抑癌基因的缺失、突变失活导致细胞增殖失控而恶性转化,可能是肿瘤发生的分子生物学基础。随着新的治疗理念的提出和综合治疗的实施,乳腺癌的治疗水平不断提高,总生存率也在逐渐提高,但复发和转移仍是乳腺癌治疗失败和死亡的主要原因,因此研究乳腺癌生物学行为,寻找治疗敏感性相关生物学标志物将有助于临床工作。HIF-1α和Her-2是乳腺癌两个独立的预后因子,对乳腺癌的发生、发展和转移有重要影响,在乳腺癌中的生物学作用以及作为提示乳腺癌的预后因素和针对其研制特异性的单克隆抗体来治疗乳腺癌日益成为近年来乳腺癌研究的热点。但以往国内外研究多见于免疫组化对乳腺癌的研究,免疫组化研究操作比较复杂、耗时、费力,而且不能对患者进行实时、动态检测病情变化,目前对HIF-1α和Her-2ECD在乳腺癌血清中的联合定量检测尚无相关报导。甘氨酸、丙氨酸作为肿瘤细胞重要的合成原料和代谢产物,受HIF-1α通道调控,在肿瘤的发生、发展和转归都有举足轻重的作用,但相关研究亦未见报道,故寻找一种新的检测方法,定量分析人体代谢途径中相关的肿瘤标志物,尽可能提高肿瘤标志物的检出率是非常重要的。本课题利用悬浮芯片技术联合高效液相色谱定量检测乳腺浸润性导管癌患者血清中HIF-1α、Her-2ECD、Ala和Gly含量变化规律,分析其内在的相关性及其与乳腺癌临床病理、生物学特性的关系；探讨HIF-1α、Her-2ECD、Ala和Gly在乳腺癌诊断和监测病情的作用,以及评估其对乳腺癌诊断的价值。
目的
本研究拟从乳腺癌患者机体的代谢途径寻找动态的肿瘤标志物,探讨他们之间内在相关性。研究采用悬浮芯片联合高效液相色谱技术,分析HIF-1α、Her.2ECD、甘氨酸和丙氨酸的含量变化,及其与临床病理因素和预后的关系,并进一步探索HIF-1α、HER-2ECD、Ala和Gly在乳腺癌诊断、预后评估和疗效评价中的价值和意义。
方法
1.病例来源选自宁波大学医学院附属医院乳腺外科2006-2008年住院病人,其中乳腺原发浸润性导管癌117例,术后复发乳腺浸润导管癌10例,乳腺良性肿癌20例,体检健康拟行乳腺美容手术31例(正常对照)。乳腺原发浸润性导管癌术前均未进行相关放疗、化疗、内分泌治疗及免疫治疗。
2.血清标本采集所有入选对象在清晨空腹采肘正中静脉血10ml,静置120分钟,3000r/min离心20分钟取血清,编号,-80℃冰箱保存,待检(根据术后的病理结果筛选保存的乳腺浸润性导管癌和良性肿瘤及乳腺美容患者血清标本冷藏待检)。
3.组织标本的采集术中将手术切除的恶性肿瘤或良性肿瘤标本切取无坏死的瘤组织各约1g,以及在行乳腺美容手术时切取正常组织约1g,然后用冰盐水冲洗除掉血污,立即置入液氮中,保存于-80℃低温冰箱。检测前分别取少许肿瘤组织,称重后用质量分数为0.9%的冰冷生理盐水按150mg:1.5ml的比例在冰浴下超声粉碎制成10%的肿瘤组织匀浆,以4000r/min(离心半径8cm)离心10min,取上清液冷藏保存待检测。
4.检测方法血清HIF-1α和Her-2ECD含量采用悬浮芯片试剂盒检测(检测范围>1pg),并用酶联免疫吸附(ELISA)法测定做对照(HIF-1α检测范围：10000-156pg;Her-2ECD检测范围：>0.39ng/ml)。血清和组织中甘氨酸和丙氨酸利用高效液相色谱法检测,利用配套软件计算出含量。
5.统计学分析采用SPSS13.0软件进行,所有数据以均数±标准差(x±s)表示,多组间比较采用方差分析(F检验),组间两两比较采用t检验,P<0.05为差异,有统计学意义。对液相芯片法与ELISA或HPLC的检测结果采用Spearson积矩相关分析,检验水准,a=0.05。
结果
1.临床病例资料：本研究四组共178例,均为女性,年龄分布为27-91岁,中位年龄49.2岁。乳腺原发浸润性导管癌117例,年龄27-91岁(平均51.56岁)；术后复发乳腺浸润导管癌10例,年龄35-57岁(平均47.6岁)；乳腺良性肿瘤20例,年龄40-63岁(平均50.38岁)；乳腺美容31例(正常对照),年龄27-65岁(平均48.77岁)。四组入选病人年龄没有统计学差异(P>0.05)。乳腺原发浸润性导管癌术前均未进行相关放疗、化疗、内分泌治疗及免疫治疗,临床病理分期：Ⅰ期25例(21.5%)、Ⅱ期61例(52.1%)、Ⅲ期19例(16.2%)、Ⅳ期12例(10.3%)；病理组织学分级：G1期12例(10.3%)、G2期41例(35.0%)、G3期64例(54.7%)；肿瘤大小：T1组<2cm 41例(35.0%)、T2组>2cm,≤5cm 61例(52.1%)、T3组>5cm15例(12.8%)；淋巴结转移情况：无转移69例(59.0%)、转移48例(41%)。
2.悬浮芯片法检测结果
2.1原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组和正常对照组血清HIF-1α含量组间有统计学差异(F=200.035,p=0.000)；复发浸润导管癌组血清HIF-1α含量(231.72±38.39)高于原发浸润导管癌(166.18±59.15)(P<0.05)；良性肿瘤组(47.77±15.26)和正常对照组(47.84±11.36)血清中HIF-1α含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清HIF-1α含量均高于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清HIF-1α含量也均高于良性肿瘤组及正常对照组(P<0.01)；
原发乳腺浸润性导管组血清HIF-1水平含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=15.030,p=0.000)：Ⅰ期(150.96+38.85)、Ⅱ期(149.52±52.96)之间没有统计学差异(P>0.05)；Ⅲ期(189.03±60.36)、Ⅳ期(249.54±44.84)之间有统计学差异(P<0.01)；Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)；Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)：发乳腺浸润性导管癌组血清HIF-1水平含量在组织学分级中组间有统计学差异(F=18.772,p=0.000)：随低分化(182.42±61.79)、中分化(154.39±52.67)和高分化(119.80±23.28)组织分化程度升高其含量逐渐减低,有统计学差异(P<0.01)；肿瘤直径大于5cm(T3)其血清HIF-1水平含量(215.19±57.11),高于T1(152.42±41.05)、T2(161.82±61.69)两组,有统计学差异(P<0.01),但T1组、T2组没有统计学差异(P>0.05)。淋巴结转移组血清HIF-1水平含量(197.48±55.28)高于没有淋巴结转移组(144.40±51.80),有统计学差异(P<0.01),孕激素受体阴性表达组(186.16±59.35)高于阳性表达组(153.23±55.69),有统计学差异(P<0.01)；雌激素受体阳性表达组(159.91±56.41)与阴性表达组(179.20±63.27)之间没有统计学差异(P>0.05)。
2.2原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组和正常对照组血清Her-2ECD含量组间有统计学差异(F=50.924,p=0.000)；复发浸润导管癌组血清Her-2ECD含量(17.61±3.63)高于原发浸润导管癌(11.20±6.116)(P＜0.05)；良性肿瘤组(4.91±3.40)和正常对照组(4.56±2.95)血清中Her-2ECD含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Her-2ECD含量均高于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Her-2ECD含量也均高于良性肿瘤组及正常对照组(P<0.01)；
原发乳腺浸润性导管组血清Her-2ECD水平含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=71.912,p=0.000)；Ⅰ期(7.89±2.94)与Ⅱ期(9.30±4.48)之间没有统计学差异(P>0.05)；Ⅲ期(15.44±6.38)与Ⅳ期(21.67±2.76)之间有统计学差异(P＜0.01)：Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)；Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)；发乳腺浸润性导管癌组血清Her-2ECD水平含量在组织学分级中组间有统计学差异(F=18.086,p=0.000)：肿瘤组织低分化组的Her-2ECD含量(13.04±6.53)高于中分化组(9.33±5.33)和高分化组(7.80±1.12),有统计学差异(P<0.01)中分化组与高分化组之间没有统计学差异(P=0.251)：Her-2ECD水平含量随T1组(7.98±3.35)、T2组(11.17±6.03)、T：组(18.02±5.57)肿瘤直径增大而含量增高(T3>T：>T1),有统计学差异(P<0.01)；淋巴结转移组Her-2ECD的含量(15.75±7.06)高于没有淋巴结转移组(8.04±2.15),有统计学差异(P<0.01)；雌激素受体阴性表达组血清Her-2ECD水平(13.49±6.43)高于雌激素受体阳性表达组(10.10±5.68),有统计学差异(P<0.01)。孕激素受体阴性表达组的Her-2ECD水平(13.05±6.42)高于孕激素受体阳性表达组(10.01±5.64),有统计学差异(P＜0.01)。
3.高效液相色谱法检测结果
3.1原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组及正常对照组血清中Ala含量组间有统计学差异(F=20.635,p=0.000)；复发浸润导管癌组血清中Ala含量(240.71±74.33)低于原发浸润导管癌(338.57±97.37)(P<0.05)；良性肿瘤组(414.49±64.94)和正常对照组(423.37±80.57)血清中Ala含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Ala含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Ala含量也均低于良性肿瘤组及正常对照组(P<0.01)
原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组及正常对照组血清中Gly含量组间有统计学差异(F=150.986,p=0.000)；复发浸润导管癌组血清中Gly含量(135.49±31.34)低于原发浸润导管癌(176.06±46.96)(P＜0.01)良性肿癌组(320.22±42.84)和正常对照组(342.19±48.15)血清中Gly含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Gly含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Gly含量也均低于良性肿癌组及正常对照组(P<0.01)
原发浸润性导管癌、复发浸润性导管癌、良性肿癌组及正常对照组组织中Ala含量组间有统计学差异(F=161.011,p=0.000)：复发浸润导管癌组织中Ala含量(224.62±44.96)低于原发浸润导管癌(177.14±67.99)(P<0.05)；良性肿瘤组(437.44±58.10)和正常对照组(406.50±66.85)组织中Ala含量之间没有统计学意义(P>0.05)。原发浸润性导管癌组织Ala含量均低于良性肿瘤组及正常对照组(P<0.01)：复发浸润性导管癌组织Ala含量也均低于良性肿瘤组及正常对照组(P<0.01)。
原发浸润性导管癌含量、复发浸润性导管癌、良性肿瘤组及正常对照组组织中Gly含量组间有统计学差异(F=141.398,p=0.000)；复发浸润导管癌组组织中Gly含量(204.64±40.94)低于原发浸润导管癌(147.92±56.74)(P＜0.01)；良性肿瘤组(327.32±46.93)和正常对照组(338.06±49.60)组织中Gly含量之间没有统计学意义(P>0.05)。原发浸润性导管癌组织Gly含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌组织Gly含量也均低于良性肿瘤组及正常对照组(P<0.01)。
3.2原发乳腺浸润性导管组血清Ala含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=32.863,p=0.000)；Ⅰ期(433.51±67.69)、Ⅱ(342.90±74.78)、Ⅲ期(272.42±60.76)及Ⅳ期(210.05±82.32)随临床病理分期增高含量逐渐降低,有统计学差异(P<0.01)；原发乳腺浸润性导管癌组血清Ala含量在组织学分级中组间有统计学差异(F=5.511,p=0.005)；低分化(389.91±76.27)、中分化(362.99±92.43)和高分化(313.29±97.36)三组随肿瘤组织分化程度升高而含量水平逐渐下降,均有统计学差异(P<0.01)；原发乳腺浸润性导管组血清Ala含量在不同大小肿瘤组之间有统计学差异(F=51.067,p=0.000)；T1组(421.19±75.45)、T2组(323.10±87.08)、T3组(241.02±43.87)随肿瘤体积增大含量逐渐减少,有统计学差异(P<0.01)；淋巴结转移组血清Ala水平(297.72±96.01)低于没有发生转移组的水平(366.98±88.35),有统计学差异(P<0.01)：雌激素受体的阳性表达组(343.37±90.31)和阴性表达组(328.58±111.27)孕激素受体的阳性表达组(349.52±96.23)与阴性表达组(321.67±97.75)的血清Ala水平之间没有统计学意义(P>0.05)。
3.3原发乳腺浸润性导管组血清Gly含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=24.621,p=0.000)；Ⅰ期(202.88±37.91)与Ⅱ期(186.18±36.57之间没有统计学差异(P>0.05)；Ⅲ期(152.54±30.75)与Ⅳ期(101.78±45.21)之间有统计学差异(P<0.01)；Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)：Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)；原发乳腺浸润性导管癌组血清Gly含量在组织学分级中组间有统计学差异(F=23.602,p=0.000)；高分化组(217.50±25.89)与中分化组(199.12±39.35)之间没有统计学意义(P>0.05)；低分化组(153.520±42.089)与高、中分化组之间均有统计学差异(P<0.01)；原发乳腺浸润性导管组血清Gly含量在不同大小肿瘤组之间有统计学差异(F=10.403,p=0.000)；T1组(195.832±37.076)与T2组(177.103±45.486)没有统计学意义(P=0.081)；T1与T3组(130.260±43.177)及T2和T3(p=0.000)均有统计学差异；淋巴结转移组血清Gly水平(152.158±46.436)低于没有发生转移组的水平(192.69±39.85),有统计学差异(P<0.01)；雌激素受体的阳性表达组(181.75±42.12)和阴性表达组(164.22±54.41)、孕激素受体的阳性表达组(182.73±45.163)与阴性表达组(165.767±48.31)的血清Gly水平之间亦没有统计学差异(P>0.05)。
4. HIF-1α、Her-2ECD和Ala、Gly之间的相关性分析：Spearson积矩相关分析表明,乳腺癌血清HIF-la与Her-2ECD呈正相关(r=0.533,p=0.000)；乳腺癌血清与组织中Ala(r=-0.429,p=0.000)的含量变化呈负相关：血清与组织中Gly(r=-0.481,p=0.000)的含量变化也呈负相关；Spearman等级相关分析示：在乳腺浸润性导管癌中血清HIF-1α水平与血清Ala(r=-0.403,p=0.000)呈负相关,在血清HIF-1α水平与Gly(r=-0.413,p=0.000)呈负相关；血清Her-2ECD与血清Ala(r=-0.509,p=0.000)水平呈负相关,血清Her-2ECD与血清Gly(r=-0.481,p=0.000)也呈负相关。
5. HIF-1α(87.1%)和Gly(96.6%)对乳腺癌诊断率高于Her-2CED (69.7%)和Ala(67.4%),HIF-1α的对乳腺癌早期诊断效果高于Her-2CED(P<0.01);Her-2CED(84.6%)对乳腺癌淋巴结转移的符合率高于HIF-1α(67.5%)、Ala(64.1%)和Gly(65.8%)。Her-2CED对乳腺癌淋巴结的转移诊断效果优越HIF-1α(P<0.01)。
6.通过配对t检验及其相关性分析、判别分析及ROC曲线分析,运用悬浮芯片技术同时定量检测血清HIF-1α和Her-2ECD的能力不低于单一比对ELISA试剂盒,悬浮芯片检测结果高于对比试剂盒的检测结果,两种方法检测HIF-1α(r=0.995,p=0.000)和Her-2ECD (r=0.998, p=0.000)结果呈正相关,相关性非常密切。
结论
1.运用悬浮芯片技术联合检测血清HIF-1α和Her-2ECD两种肿瘤标志物的含量,与比对试剂盒比较的相关性好,实验结果可靠,灵敏度高、操作简单,对乳腺癌的诊断和研究具有较高的辅助价值。可用于临床检测和课题实验研究。
2.乳腺浸润性导管癌HIF-1α和Her-2ECD水平过表达,是病人预后不良的指征,其过度表达说明它在肿瘤发生、转化中起重要作用。所以动态检测乳腺浸润性导管癌血清HIF-1α和Her-2ECD水平可提供实时、动态的信息,为制定治疗方案提供帮助。因此检测血清HIF-1α和Her-2ECD水平的动态变化对乳腺浸润性导管癌复发、转移的早期判断及预后评估具有重要意义。
3.Ala和Gly参与了乳腺浸润性导管癌细胞的合成和能量代谢,在乳腺浸润性导管癌的发生、发展和转归中有重要作用,可能成为乳腺浸润性导管癌靶向治疗新的靶点。
4.血清HIF-1α、Her-2ECD、Ala和Gly可作为乳腺癌的早期诊断、评价乳癌恶性程度和预后比较全面的指标,能准确的反映乳腺浸润性导管癌的生物学特性,随着对血清Her-2ECD、HIF-1α、Ala和Gly基础和临床研究深入以其为靶点的治疗可能成为肿瘤治疗和预防的重要手段之一。
Background
Breast cancer is one of the most common malignancies among women around the world, seriously threatening women's physical and mental health. The incidence of breast cancer around the world has shown a steady upward trend, in the late 20th century the world's annual statistics has show that about 1.15 million people are diagnosed with breast cancer,410 thousand people die of this disease, and the patients are younger and younger. Breast cancer has became the leading cause of death among female patients. However, the etiology of breast cancer is not yet clear, its pathogenesis is very complex and many factors affect the incidence. In recent years, with the deepening of research in tumor molecular pathology and molecular biology, it is recognized that the occurrence and development of cancer is a gradual process involving multi-stage and multi-gene changes. A number of oncogene proteins encoded by the abnormal activation, over-expression and deletion of tumor suppressor genes, and malignant transformation caused by inactivating mutations and uncontrolled cell proliferation may be the molecular biological basis of tumor. New ideas of treatment put forward and the implementation of comprehensive treatment, the treatment of breast cancer has improved continuously. The overall survival rate is gradually increased, but recurrence and metastasis of breast cancer are still the main causes for treatment failure and death. Therefore, the study of biological behavior in breast cancer, and discovering the relevant biological markers of sensitivity will contribute to clinical work. HIF-1αand Her-2ECD are two independent prognostic factors of breast cancer, which have significant influence on the incidence of, development and metastasis of breast cancer. Due to their biological roles in breast cancer, they are implications of prognostic factors of breast cancer and have important functions in specific monoclonal antibodies to treat breast cancer, HIF-1αand Her-2ECD have increasingly become hot research issues in recent years. In the past domestic and foreign researches prevalent in the immunohistochemical study of breast cancer, immunohistochemical study of operation is relatively more complex, time-consuming, laborious, and can not detect the real-time and dynamic changes in patients, and there is no relevant reports about co-quantitative detection of HIF-1αand Her-2ECD in the serum of breast cancer patients. Glycine and alanine, regulated by HIF-1αchannel, which are important synthetic raw materials of tumor cells and metabolic products of tumor, play very pivotal roles in the occurrence, development and metastasis of the cancer, but no related research has ever been reported. So it is very important to look for a new detection method, quantitatively analyzing the tumor markers of related metabolic pathways, to increase the detection rate to the maximum.
This research subject is to apply floating-chip technology combining with high-performance liquid chromatography with quantitative detection of change in content of HIF-1α, Her-2ECD, Ala and Gly content in breast carcinoma patients, analzing its relevance and its relationship with the internal breast cancer pathology, and the relationship between the biological characteristics, and approach the role of HIF-la, Her-2ECD, Ala and Gly in breast cancer diagnosis and monitoring of illness, as well as to assess its diagnostic value of breast cancer.
Objectives
This study is based on chip technology combined with high-performance liquid chromatography suspended from the body's metabolic pathways of breast cancer patients to find the dynamic tumor markers to explore the intrinsic correlation between them, to analyze HIF-1α, Her-2ECD, glycine and alanine acid changes and clinical pathological factors and prognosis and study the prognosis of breast cancer. And to further explore the value and significance of her-2ECD, HIF-la, Ala and Gly in the diagnosis of breast cancer.
Methods
1. Cases from Breast surgery of Ningbo University School of Medicine Affiliated Hospital from 2006 to 2008, Primary breast infiltrating ductal carcinoma:117 cases. Postoperative recurrence of breast infiltrating ductal carcinoma:10 cases. Benign breast tumor:20 cases. breast cosmetic surgery of the health:31 cases(The normal control group). Primary invasive ductal breast cancer were not treated by preoperative radiotherapy, chemotherapy, endocrine therapy and immunotherapy.
2. Serum specimen collection:with the patient's own consent, each selected object is collected blood 10ml in median cubital vein in the early morning. Put them aside for 120 minutes, then take serum after 3000r/min decentralization for 20 minutes, number, and preserve in -80℃refrigerator, screening serum of the breast invasive ductal carcinoma and benign tumor and breast cosmetic surgery of the health patients for inspection according to surgery pathological findings.
3. The collection of tissue samples:with the consent of selected cancer patients, in which surgery would be surgical resection of malignant or benign tumor specimens were cut without necrosis of tumor tissue, each about 1g, and the removal of breast cosmetic surgery at the same time normal tissue of about 1g, and then rinse off bloody with ice saline, immediately placed in liquid nitrogen and stored at -80℃low temperature refrigerators. Before test, take some tumor tissues, respectively, and uonicate to 10% homogenatation under the ice bath after weighing with the ice-cold 0.9% saline by the ratio of 150mg:1.5ml,4000r/min (centrifugal radius 8cm) centrifuge for 10min.The supernatant stored frozen for testing.
4. Sample detection:Serum HIF-1αand Her-2ECD content by suspension chip detection kit (detection range>1pg), and enzyme-linked immunosorbent assay (ELISA) is determinated as controls (HIF-la detection range:10000-156pg; Her-2ECD detection range:> 0.39 ng/ml). Serum and tissues of glycine and alanine are tested by high-performance liquid chromatography, using software packages to calculate concentration.
5. Statistical analysis:SPSS13.0 data were used, all data are mean±standard deviation (x±s). Use analysis of variance (F test)to compare between the two groups. T test was used to compare among two groups, P<0.05 for the difference, there were statistical significances. On the liquid-phase ELISA or HPLC-chip method and the results of tests were using Spearson rank correlation analysis. Test level,α=0.05.
Results
1. Clinical Case Data:178 cases in this group were female, age distribution from 27 to 91 years old, with a median age of 49.2 years old. Primary invasive ductal breast carcinoma of 117 patients, aged from 27 to 91 years old (mean 51.56 years); postoperative recurrence of breast invasive ductal carcinoma of 10 cases, from 35 to 57 years old (mean 47.6 years); 20 cases of benign breast tumor, age 40 to 63 years old (mean 50.38 years); breast cosmetic surgery of 31 cases(The normal control group), aged from 27 to 65 years old (mean 48.77 years); Four groups selected for the patient's age were not statistically significant (P>0.05), All women.Primary invasive ductal breast cancer were not associated preoperative radiotherapy, chemotherapy, endocrine therapy and immunotherapy, clinical pathological stage:Ⅰperiod of 25 cases (21.5%),Ⅱperiod of 61 cases (52.1%),Ⅲperiod of 19 cases (16.2%);Ⅳ,12 cases (10.3%) histopathological grading:G1 phase 12 cases (10.3%), G2 phase 41 cases (35.0%), G3 period of 64 cases (54.7%); tumor size: T1 group≤2cm 41 Li (35.0%), T2 Group> 2cm,≤5cm 61 Li (52.1%), T3 group> 5cm15 cases (12.8%); lymph node metastasis:69 cases without metastasis (59.0%), the transfer of 48 patients (41%).
2. Results of suspension micro-array test
2.1 Primary invasive ductal carcinoma (166.18±59.15) and recurrent invasive ductal carcinoma (231.72±38.39) of serum levels of HIF-1αwere higher than benign tumor group (47.77±15.26) and normal control group (47.84±11.36) (P<0.01); Recurrent infiltrating ductal carcinoma of HIF-1αserum levels were significantly higher than those in primary invasive ductal carcinoma of (P<0.01和P<0.05); There were not statistically significant between the benign tumor group and normal control group serum and tissue HIF-1αlevels. Primary invasive ductal carcinoma of serum levels of HIF-1 levels in clinical and pathological staging of one (150.96±38.85), between the two groups (149.52±52.96) there were no statistical significance(P>0.05), but there were a significant difference between three (189.03±60.36), four stages (249.54±44.84) and one, two stages (P<0.01); and with reduce histological grade (well-differentiated, moderately differentiated and poorly differentiated), its content gradually elevated (119.80±23.28 154.39±52.67,182.42±61.79), there were a significant difference (P<0.01); tumor when the diameter of T3 (diameter>5cm), the serum levels of HIF-1 were significantly higher (215.19±57.11) than the other two groups (T1,T2) (152.42±41.05,161.82±61.69), there were a significant difference (P<0.01); Tumor diameter, T1, T2 were not statistically significant (P>0.05). Lymph node metastasis in patients with serum levels of HIF-1 were significantly higher (197.48±55.28) than that in patients with lymph node metastasis did not occur (144.40±51.80), there were a significant difference(P<0.01), progesterone receptor-negative expression (186.16±59.35) was significantly higher than positive expression(153.23±55.69), there were a significant difference (P<0.01); expression of estrogen receptor-positive (159.91±56.41) and negative expression (179.20±63.27) had no significant difference (P>0.05).
2.2 Primary invasive ductal carcinoma (11.20±6.116) and invasive ductal carcinoma of recurrence (17.61±3.63) of serum Her-2ECD levels were significantly higher than benign (4.91±3.40) and normal control group (4.56±2.95) (P<0.01), Recurrent infiltrating ductal carcinoma higher than primary invasive ductal carcinoma (P<0.01); There were no statistical significance between the benign tumor group and normal control serum Her-2ECD content(P>0.05).Primary invasive ductal breast cancer patients with Her-2ECD level of content and serum levels of HIF-1 were similar, to the clinical pathological staging one(7.89±2.94), two (9.30±4.48), there were no statistically significant (P>0.05), but there is a significant difference between three (15.44±6.38), four stages (21.67±2.76) and one, two stages (P<0.01); tumor tissue with low (13.04±6.53) credit-based were higher than those higher degree of differentiation (in the differentiation and well-differentiated) (9.33±5.33,7.80±1.12), there were a significant difference (P<0.01); Her-2ECD level of concentration with the increase of tumor diameter for increased levels (T3>T2>T1) (18.02±5.57,11.17±6.03,7.98±3.35), there is a significant difference (P<0.01); Lymph node metastasis (15.75±7.06) than those without metastasis (8.04±2.15) in patients with serum Her-2ECD levels were significantly increased, there were a significant difference (P<0.01); Estrogen and progesterone receptor positive and negative expression of Her-2ECD between the serum levels were different, and negative expression was significantly higher (13.49±6.43,13.05±6.42) than positive expression (10.10±5.68,10.01±5.64), there is a significant difference (P<0.01).
3. Results of high-performance liquid chromatography
3.1 Analysis found that serum Ala (338.57±97.37), Gly (176.06±46.9) content in the primary, recurrent invasive ductal carcinoma (240.71±74.33,135.49±31.34) were lower than the normal benign tumor group (414.49±64.94,320.22±42.84) and control group (423.37±80.57,342.19±48.15) (P<0.01); That tissues Ala (177.14±67.99), Gly (147.92±56.74) content in the primary, recurrent invasive ductal carcinoma (224.62±44.96,204.64±40.94) were lower than the normal benign tumor group (437.44±58.10,327.32±46.93) and control group too (406.50±66.85,338.06±49.60) (P<0.01);Ala, Gly in recurrent infiltrating ductal carcinoma group were significantly higher than those in primary invasive ductal carcinoma (P<0.01和P<0.05); While the Ala, Gly in the serum of recurrent infiltrating ductal carcinoma were significantly lower than that of primary invasive ductal carcinoma (P<0.01和P<0.05); Ala, Gly levels in serum and tissues were no statistical significance between the benign tumor group and normal control group.
3.2 Primary invasive ductal carcinoma of the level of serum Ala levels reduce with the clinical and pathological stage gradually increase (433.51±67.69, 342.90±74.78,272.42±60.76,210.05±82.32), there were a significant difference between the levels of different stages (P<0.01); With the histological grade (pathology classification) increased, the level of serum Ala levels reduce (389.91±76.27,362.99±92.43,313.29±97.36), there were a significant difference between different groups (P<0.01); Serum Ala levels and tumor volume is also closely related to tumor volume increased with decreasing concentration(421.19±75.45,323.10±87.08,241.02±43.87), there are significant differences. And lymph node metastases were also inter-related occurred in patients, with lymph node metastasis (297.72±96.01) in Ala serum levels were significantly lower than the transfer did not occur (366.98±88.35) in patients with the level, there were a significant difference (P<0.01); Estrogen (343.37±90.31,328.58±111.27) and progesterone (349.52±96.23 321.67±97.75) receptors and serum in Ala levels were not statistically significant (P>0.05).
3.3 As to primary invasive ductal carcinoma of Gly serum levels in clinical and pathological staging, there were no significant difference betweenⅠ(202.88±37.91),Ⅱperiod (186.18±36.57) (P>0.05), there were a significant difference betweenⅠ,Ⅱperiod andⅢ(152.54±30.75),Ⅳperiod (101.78±45.21) (P<0.01), its content reduced gradually with the classification increased; In tumor cell differentiation, there were no statistical significance between the well-differentiated group (217.50±25.89) and moderately differentiation group (199.12±39.35) (P>0.05), there were statistically significant between the well-differentiated group, moderately differentiation group and poorly differentiation group (153.520±42.089) (P<0.01), with the reduction in the degree of differentiation Gly levels were gradually reduced; Gly serum levels and tumor volume were also closely related to tumor volume increased with decreasing concentration (195.832±37.076, 177.103±45.486,130.260±43.177), there are significant differences(P<0.01). And lymph node metastases were also inter-related occurred in patients with lymph node metastasis, in the Gly serum levels (152.158±46.436) were significantly lower than the transfer did not occur in patients with the level (192.69±39.85), there were a significant difference (P<0.01); Estrogen (181.75±42.12,164.22±54.41) and progesterone receptors (182.73±45.163, 165.767±48.31) and serum of Gly levels were not statistically significant (P> 0.05).
4. Correlation between HIF-1α, Her-2ECD and Ala, Gly:Spearson rank correlation analysis showed that HIF-1αwere positive correlation with Her-2ECD in serum (r=0.533,p=0.000);Ala, (r=-0.429, p=0.000) Gly (r=-0.481, p=0.000) content was negatively correlated with changes in breast cancer serum and tissue. It was closely negatively correlated between HIF-1α, Her-2ECD and Ala (r=-0.403, r=-0.413, p=0.000), Gly (r=-0.509, r=-0.481, p=0.000) in breast invasive ductal carcinoma serum, and showed a closely positive correlation with Ala (r=0.973, r=0.969, p=0.000), Gly (r=0.534, r=0.533, p=0.000) in organization. There were no statistically significance between the normal control group and benign tumors(P>0.05).
5. HIF-1α(87.1%) and Gly(96.6%) is higher than Her-2CED(69.7%) and Ala(67.4%). in diagnosis on breast cancer. Early diagnosis on breast of HIF-1αis higher than Her-2CED(P<0.01); Her-2CED(84.6%) is higher than HIF-1α(67.5%), Ala(64.1%) and Gly(65.8%) in lymph node metastasis of breast cancer. Her-2CED is superior to HIF-1α(P<0.01) in lymph node metastasis of breast cancer.
6. Through the correlation analysis, discriminant analysis and ROC curve analysis, suspended chip technology the ability to detect a single target goal of not less than ELISA kit, test results and contrast the results of tests kits was no significant difference, especially for the determination of the value of tumor samples, suspended Chip kit and the control kit relatively good correlation (r=0.995, r= 0.998, p=0.000)。
Conclusion
1. In serum, HIF-1αand Her-2ECD can be detected with high sensitivity, high-throughput and reliability by using Suspension Array Technology which had a better auxiliary value than ELISA kit in the diagnosis of breast cancer. Therefore, this theme had a viable method and a reliable result, that can be used for research project.
2. The lever of HIF-1αand Her-2ECD in serum of breast invasive ductal carcinoma were overexpression, it were an indication of poor prognosis of the patients, its overexpression indicates that it played an important role in tumorigenesis and tumor induction. So dynamic detection of HIF-1αand Her-2ECD can be used as a real-time, dynamic information, to provide assistance for the development of making treatment program. Therefore, it was important for us to detect the level of HIF-1αand Her-2ECD in serum in the recurrence, early assessment of metastasis and prognosis of breast invasive ductal carcinoma.
3. Ala and Gly involved in anabolic and energy metabolism of the tumor cells of the breast invasive ductal carcinoma, played an important role in the occurrence, development and progression of the breast invasive ductal carcinoma, and may become a new target of the targeted therapy.
4. In serum, HIF-1α、Her-2ECD、Ala and Gly can be used as the early diagnosis of breast cancer, some more comprehensive indicators to evaluate the degree of malignancy, can reflect the biological characteristics of the breast invasive ductal carcinoma. With the basic and clinical research of HIF-1α、Her-2ECD、Ala and Gly in serum going further, it could be one of means in cancer treatment and prevention for targeted treatment.
乳腺癌是全世界女性最常见的恶性肿瘤之一,严重威胁女性的身心健康。全世界乳腺癌的发病率一直呈稳步上升的趋势,20世纪末的统计资料表明世界每年约有115万人诊断为乳腺癌,有41万人死于该病,发病年龄也有年轻化趋势,是导致女性肿瘤患者死亡的第一大原因。然而,乳腺癌的病因尚不十分清楚,其发病机制也非常复杂,影响发病的因素很多。近年来,随着肿瘤分子病理学和分子生物学研究的深入,人们认识到肿瘤的发生和发展是一个多阶段、多基因改变参与的渐进过程。多个癌基因的异常激活和编码蛋白的过度表达以及抑癌基因的缺失、突变失活导致细胞增殖失控而恶性转化,可能是肿瘤发生的分子生物学基础。随着新的治疗理念的提出和综合治疗的实施,乳腺癌的治疗水平不断提高,总生存率也在逐渐提高,但复发和转移仍是乳腺癌治疗失败和死亡的主要原因,因此研究乳腺癌生物学行为,寻找治疗敏感性相关生物学标志物将有助于临床工作。HIF-1α和Her-2是乳腺癌两个独立的预后因子,对乳腺癌的发生、发展和转移有重要影响,在乳腺癌中的生物学作用以及作为提示乳腺癌的预后因素和针对其研制特异性的单克隆抗体来治疗乳腺癌日益成为近年来乳腺癌研究的热点。但以往国内外研究多见于免疫组化对乳腺癌的研究,免疫组化研究操作比较复杂、耗时、费力,而且不能对患者进行实时、动态检测病情变化,目前对HIF-1α和Her-2ECD在乳腺癌血清中的联合定量检测尚无相关报导。甘氨酸、丙氨酸作为肿瘤细胞重要的合成原料和代谢产物,受HIF-1α通道调控,在肿瘤的发生、发展和转归都有举足轻重的作用,但相关研究亦未见报道,故寻找一种新的检测方法,定量分析人体代谢途径中相关的肿瘤标志物,尽可能提高肿瘤标志物的检出率是非常重要的。本课题利用悬浮芯片技术联合高效液相色谱定量检测乳腺浸润性导管癌患者血清中HIF-1α、Her-2ECD、Ala和Gly含量变化规律,分析其内在的相关性及其与乳腺癌临床病理、生物学特性的关系；探讨HIF-1α、Her-2ECD、Ala和Gly在乳腺癌诊断和监测病情的作用,以及评估其对乳腺癌诊断的价值。
目的
本研究拟从乳腺癌患者机体的代谢途径寻找动态的肿瘤标志物,探讨他们之间内在相关性。研究采用悬浮芯片联合高效液相色谱技术,分析HIF-1α、Her.2ECD、甘氨酸和丙氨酸的含量变化,及其与临床病理因素和预后的关系,并进一步探索HIF-1α、HER-2ECD、Ala和Gly在乳腺癌诊断、预后评估和疗效评价中的价值和意义。
方法
1.病例来源选自宁波大学医学院附属医院乳腺外科2006-2008年住院病人,其中乳腺原发浸润性导管癌117例,术后复发乳腺浸润导管癌10例,乳腺良性肿癌20例,体检健康拟行乳腺美容手术31例(正常对照)。乳腺原发浸润性导管癌术前均未进行相关放疗、化疗、内分泌治疗及免疫治疗。
2.血清标本采集所有入选对象在清晨空腹采肘正中静脉血10ml,静置120分钟,3000r/min离心20分钟取血清,编号,-80℃冰箱保存,待检(根据术后的病理结果筛选保存的乳腺浸润性导管癌和良性肿瘤及乳腺美容患者血清标本冷藏待检)。
3.组织标本的采集术中将手术切除的恶性肿瘤或良性肿瘤标本切取无坏死的瘤组织各约1g,以及在行乳腺美容手术时切取正常组织约1g,然后用冰盐水冲洗除掉血污,立即置入液氮中,保存于-80℃低温冰箱。检测前分别取少许肿瘤组织,称重后用质量分数为0.9%的冰冷生理盐水按150mg:1.5ml的比例在冰浴下超声粉碎制成10%的肿瘤组织匀浆,以4000r/min(离心半径8cm)离心10min,取上清液冷藏保存待检测。
4.检测方法血清HIF-1α和Her-2ECD含量采用悬浮芯片试剂盒检测(检测范围>1pg),并用酶联免疫吸附(ELISA)法测定做对照(HIF-1α检测范围：10000-156pg;Her-2ECD检测范围：>0.39ng/ml)。血清和组织中甘氨酸和丙氨酸利用高效液相色谱法检测,利用配套软件计算出含量。
5.统计学分析采用SPSS13.0软件进行,所有数据以均数±标准差(x±s)表示,多组间比较采用方差分析(F检验),组间两两比较采用t检验,P<0.05为差异,有统计学意义。对液相芯片法与ELISA或HPLC的检测结果采用Spearson积矩相关分析,检验水准,a=0.05。
结果
1.临床病例资料：本研究四组共178例,均为女性,年龄分布为27-91岁,中位年龄49.2岁。乳腺原发浸润性导管癌117例,年龄27-91岁(平均51.56岁)；术后复发乳腺浸润导管癌10例,年龄35-57岁(平均47.6岁)；乳腺良性肿瘤20例,年龄40-63岁(平均50.38岁)；乳腺美容31例(正常对照),年龄27-65岁(平均48.77岁)。四组入选病人年龄没有统计学差异(P>0.05)。乳腺原发浸润性导管癌术前均未进行相关放疗、化疗、内分泌治疗及免疫治疗,临床病理分期：Ⅰ期25例(21.5%)、Ⅱ期61例(52.1%)、Ⅲ期19例(16.2%)、Ⅳ期12例(10.3%)；病理组织学分级：G1期12例(10.3%)、G2期41例(35.0%)、G3期64例(54.7%)；肿瘤大小：T1组<2cm 41例(35.0%)、T2组>2cm,≤5cm 61例(52.1%)、T3组>5cm15例(12.8%)；淋巴结转移情况：无转移69例(59.0%)、转移48例(41%)。
2.悬浮芯片法检测结果
2.1原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组和正常对照组血清HIF-1α含量组间有统计学差异(F=200.035,p=0.000)；复发浸润导管癌组血清HIF-1α含量(231.72±38.39)高于原发浸润导管癌(166.18±59.15)(P<0.05)；良性肿瘤组(47.77±15.26)和正常对照组(47.84±11.36)血清中HIF-1α含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清HIF-1α含量均高于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清HIF-1α含量也均高于良性肿瘤组及正常对照组(P<0.01)；
原发乳腺浸润性导管组血清HIF-1水平含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=15.030,p=0.000)：Ⅰ期(150.96+38.85)、Ⅱ期(149.52±52.96)之间没有统计学差异(P>0.05)；Ⅲ期(189.03±60.36)、Ⅳ期(249.54±44.84)之间有统计学差异(P<0.01)；Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)；Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)：发乳腺浸润性导管癌组血清HIF-1水平含量在组织学分级中组间有统计学差异(F=18.772,p=0.000)：随低分化(182.42±61.79)、中分化(154.39±52.67)和高分化(119.80±23.28)组织分化程度升高其含量逐渐减低,有统计学差异(P<0.01)；肿瘤直径大于5cm(T3)其血清HIF-1水平含量(215.19±57.11),高于T1(152.42±41.05)、T2(161.82±61.69)两组,有统计学差异(P<0.01),但T1组、T2组没有统计学差异(P>0.05)。淋巴结转移组血清HIF-1水平含量(197.48±55.28)高于没有淋巴结转移组(144.40±51.80),有统计学差异(P<0.01),孕激素受体阴性表达组(186.16±59.35)高于阳性表达组(153.23±55.69),有统计学差异(P<0.01)；雌激素受体阳性表达组(159.91±56.41)与阴性表达组(179.20±63.27)之间没有统计学差异(P>0.05)。
2.2原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组和正常对照组血清Her-2ECD含量组间有统计学差异(F=50.924,p=0.000)；复发浸润导管癌组血清Her-2ECD含量(17.61±3.63)高于原发浸润导管癌(11.20±6.116)(P＜0.05)；良性肿瘤组(4.91±3.40)和正常对照组(4.56±2.95)血清中Her-2ECD含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Her-2ECD含量均高于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Her-2ECD含量也均高于良性肿瘤组及正常对照组(P<0.01)；
原发乳腺浸润性导管组血清Her-2ECD水平含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=71.912,p=0.000)；Ⅰ期(7.89±2.94)与Ⅱ期(9.30±4.48)之间没有统计学差异(P>0.05)；Ⅲ期(15.44±6.38)与Ⅳ期(21.67±2.76)之间有统计学差异(P＜0.01)：Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)；Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)；发乳腺浸润性导管癌组血清Her-2ECD水平含量在组织学分级中组间有统计学差异(F=18.086,p=0.000)：肿瘤组织低分化组的Her-2ECD含量(13.04±6.53)高于中分化组(9.33±5.33)和高分化组(7.80±1.12),有统计学差异(P<0.01)中分化组与高分化组之间没有统计学差异(P=0.251)：Her-2ECD水平含量随T1组(7.98±3.35)、T2组(11.17±6.03)、T：组(18.02±5.57)肿瘤直径增大而含量增高(T3>T：>T1),有统计学差异(P<0.01)；淋巴结转移组Her-2ECD的含量(15.75±7.06)高于没有淋巴结转移组(8.04±2.15),有统计学差异(P<0.01)；雌激素受体阴性表达组血清Her-2ECD水平(13.49±6.43)高于雌激素受体阳性表达组(10.10±5.68),有统计学差异(P<0.01)。孕激素受体阴性表达组的Her-2ECD水平(13.05±6.42)高于孕激素受体阳性表达组(10.01±5.64),有统计学差异(P＜0.01)。
3.高效液相色谱法检测结果
3.1原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组及正常对照组血清中Ala含量组间有统计学差异(F=20.635,p=0.000)；复发浸润导管癌组血清中Ala含量(240.71±74.33)低于原发浸润导管癌(338.57±97.37)(P<0.05)；良性肿瘤组(414.49±64.94)和正常对照组(423.37±80.57)血清中Ala含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Ala含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Ala含量也均低于良性肿瘤组及正常对照组(P<0.01)
原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组及正常对照组血清中Gly含量组间有统计学差异(F=150.986,p=0.000)；复发浸润导管癌组血清中Gly含量(135.49±31.34)低于原发浸润导管癌(176.06±46.96)(P＜0.01)良性肿癌组(320.22±42.84)和正常对照组(342.19±48.15)血清中Gly含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Gly含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Gly含量也均低于良性肿癌组及正常对照组(P<0.01)
原发浸润性导管癌、复发浸润性导管癌、良性肿癌组及正常对照组组织中Ala含量组间有统计学差异(F=161.011,p=0.000)：复发浸润导管癌组织中Ala含量(224.62±44.96)低于原发浸润导管癌(177.14±67.99)(P<0.05)；良性肿瘤组(437.44±58.10)和正常对照组(406.50±66.85)组织中Ala含量之间没有统计学意义(P>0.05)。原发浸润性导管癌组织Ala含量均低于良性肿瘤组及正常对照组(P<0.01)：复发浸润性导管癌组织Ala含量也均低于良性肿瘤组及正常对照组(P<0.01)。
原发浸润性导管癌含量、复发浸润性导管癌、良性肿瘤组及正常对照组组织中Gly含量组间有统计学差异(F=141.398,p=0.000)；复发浸润导管癌组组织中Gly含量(204.64±40.94)低于原发浸润导管癌(147.92±56.74)(P＜0.01)；良性肿瘤组(327.32±46.93)和正常对照组(338.06±49.60)组织中Gly含量之间没有统计学意义(P>0.05)。原发浸润性导管癌组织Gly含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌组织Gly含量也均低于良性肿瘤组及正常对照组(P<0.01)。
3.2原发乳腺浸润性导管组血清Ala含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=32.863,p=0.000)；Ⅰ期(433.51±67.69)、Ⅱ(342.90±74.78)、Ⅲ期(272.42±60.76)及Ⅳ期(210.05±82.32)随临床病理分期增高含量逐渐降低,有统计学差异(P<0.01)；原发乳腺浸润性导管癌组血清Ala含量在组织学分级中组间有统计学差异(F=5.511,p=0.005)；低分化(389.91±76.27)、中分化(362.99±92.43)和高分化(313.29±97.36)三组随肿瘤组织分化程度升高而含量水平逐渐下降,均有统计学差异(P<0.01)；原发乳腺浸润性导管组血清Ala含量在不同大小肿瘤组之间有统计学差异(F=51.067,p=0.000)；T1组(421.19±75.45)、T2组(323.10±87.08)、T3组(241.02±43.87)随肿瘤体积增大含量逐渐减少,有统计学差异(P<0.01)；淋巴结转移组血清Ala水平(297.72±96.01)低于没有发生转移组的水平(366.98±88.35),有统计学差异(P<0.01)：雌激素受体的阳性表达组(343.37±90.31)和阴性表达组(328.58±111.27)孕激素受体的阳性表达组(349.52±96.23)与阴性表达组(321.67±97.75)的血清Ala水平之间没有统计学意义(P>0.05)。
3.3原发乳腺浸润性导管组血清Gly含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=24.621,p=0.000)；Ⅰ期(202.88±37.91)与Ⅱ期(186.18±36.57之间没有统计学差异(P>0.05)；Ⅲ期(152.54±30.75)与Ⅳ期(101.78±45.21)之间有统计学差异(P<0.01)；Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)：Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)；原发乳腺浸润性导管癌组血清Gly含量在组织学分级中组间有统计学差异(F=23.602,p=0.000)；高分化组(217.50±25.89)与中分化组(199.12±39.35)之间没有统计学意义(P>0.05)；低分化组(153.520±42.089)与高、中分化组之间均有统计学差异(P<0.01)；原发乳腺浸润性导管组血清Gly含量在不同大小肿瘤组之间有统计学差异(F=10.403,p=0.000)；T1组(195.832±37.076)与T2组(177.103±45.486)没有统计学意义(P=0.081)；T1与T3组(130.260±43.177)及T2和T3(p=0.000)均有统计学差异；淋巴结转移组血清Gly水平(152.158±46.436)低于没有发生转移组的水平(192.69±39.85),有统计学差异(P<0.01)；雌激素受体的阳性表达组(181.75±42.12)和阴性表达组(164.22±54.41)、孕激素受体的阳性表达组(182.73±45.163)与阴性表达组(165.767±48.31)的血清Gly水平之间亦没有统计学差异(P>0.05)。
4. HIF-1α、Her-2ECD和Ala、Gly之间的相关性分析：Spearson积矩相关分析表明,乳腺癌血清HIF-la与Her-2ECD呈正相关(r=0.533,p=0.000)；乳腺癌血清与组织中Ala(r=-0.429,p=0.000)的含量变化呈负相关：血清与组织中Gly(r=-0.481,p=0.000)的含量变化也呈负相关；Spearman等级相关分析示：在乳腺浸润性导管癌中血清HIF-1α水平与血清Ala(r=-0.403,p=0.000)呈负相关,在血清HIF-1α水平与Gly(r=-0.413,p=0.000)呈负相关；血清Her-2ECD与血清Ala(r=-0.509,p=0.000)水平呈负相关,血清Her-2ECD与血清Gly(r=-0.481,p=0.000)也呈负相关。
5. HIF-1α(87.1%)和Gly(96.6%)对乳腺癌诊断率高于Her-2CED (69.7%)和Ala(67.4%),HIF-1α的对乳腺癌早期诊断效果高于Her-2CED(P<0.01);Her-2CED(84.6%)对乳腺癌淋巴结转移的符合率高于HIF-1α(67.5%)、Ala(64.1%)和Gly(65.8%)。Her-2CED对乳腺癌淋巴结的转移诊断效果优越HIF-1α(P<0.01)。
6.通过配对t检验及其相关性分析、判别分析及ROC曲线分析,运用悬浮芯片技术同时定量检测血清HIF-1α和Her-2ECD的能力不低于单一比对ELISA试剂盒,悬浮芯片检测结果高于对比试剂盒的检测结果,两种方法检测HIF-1α(r=0.995,p=0.000)和Her-2ECD (r=0.998, p=0.000)结果呈正相关,相关性非常密切。
结论
1.运用悬浮芯片技术联合检测血清HIF-1α和Her-2ECD两种肿瘤标志物的含量,与比对试剂盒比较的相关性好,实验结果可靠,灵敏度高、操作简单,对乳腺癌的诊断和研究具有较高的辅助价值。可用于临床检测和课题实验研究。
2.乳腺浸润性导管癌HIF-1α和Her-2ECD水平过表达,是病人预后不良的指征,其过度表达说明它在肿瘤发生、转化中起重要作用。所以动态检测乳腺浸润性导管癌血清HIF-1α和Her-2ECD水平可提供实时、动态的信息,为制定治疗方案提供帮助。因此检测血清HIF-1α和Her-2ECD水平的动态变化对乳腺浸润性导管癌复发、转移的早期判断及预后评估具有重要意义。
3.Ala和Gly参与了乳腺浸润性导管癌细胞的合成和能量代谢,在乳腺浸润性导管癌的发生、发展和转归中有重要作用,可能成为乳腺浸润性导管癌靶向治疗新的靶点。
4.血清HIF-1α、Her-2ECD、Ala和Gly可作为乳腺癌的早期诊断、评价乳癌恶性程度和预后比较全面的指标,能准确的反映乳腺浸润性导管癌的生物学特性,随着对血清Her-2ECD、HIF-1α、Ala和Gly基础和临床研究深入以其为靶点的治疗可能成为肿瘤治疗和预防的重要手段之一。
Background
Breast cancer is one of the most common malignancies among women around the world, seriously threatening women's physical and mental health. The incidence of breast cancer around the world has shown a steady upward trend, in the late 20th century the world's annual statistics has show that about 1.15 million people are diagnosed with breast cancer,410 thousand people die of this disease, and the patients are younger and younger. Breast cancer has became the leading cause of death among female patients. However, the etiology of breast cancer is not yet clear, its pathogenesis is very complex and many factors affect the incidence. In recent years, with the deepening of research in tumor molecular pathology and molecular biology, it is recognized that the occurrence and development of cancer is a gradual process involving multi-stage and multi-gene changes. A number of oncogene proteins encoded by the abnormal activation, over-expression and deletion of tumor suppressor genes, and malignant transformation caused by inactivating mutations and uncontrolled cell proliferation may be the molecular biological basis of tumor. New ideas of treatment put forward and the implementation of comprehensive treatment, the treatment of breast cancer has improved continuously. The overall survival rate is gradually increased, but recurrence and metastasis of breast cancer are still the main causes for treatment failure and death. Therefore, the study of biological behavior in breast cancer, and discovering the relevant biological markers of sensitivity will contribute to clinical work. HIF-1αand Her-2ECD are two independent prognostic factors of breast cancer, which have significant influence on the incidence of, development and metastasis of breast cancer. Due to their biological roles in breast cancer, they are implications of prognostic factors of breast cancer and have important functions in specific monoclonal antibodies to treat breast cancer, HIF-1αand Her-2ECD have increasingly become hot research issues in recent years. In the past domestic and foreign researches prevalent in the immunohistochemical study of breast cancer, immunohistochemical study of operation is relatively more complex, time-consuming, laborious, and can not detect the real-time and dynamic changes in patients, and there is no relevant reports about co-quantitative detection of HIF-1αand Her-2ECD in the serum of breast cancer patients. Glycine and alanine, regulated by HIF-1αchannel, which are important synthetic raw materials of tumor cells and metabolic products of tumor, play very pivotal roles in the occurrence, development and metastasis of the cancer, but no related research has ever been reported. So it is very important to look for a new detection method, quantitatively analyzing the tumor markers of related metabolic pathways, to increase the detection rate to the maximum.
This research subject is to apply floating-chip technology combining with high-performance liquid chromatography with quantitative detection of change in content of HIF-1α, Her-2ECD, Ala and Gly content in breast carcinoma patients, analzing its relevance and its relationship with the internal breast cancer pathology, and the relationship between the biological characteristics, and approach the role of HIF-la, Her-2ECD, Ala and Gly in breast cancer diagnosis and monitoring of illness, as well as to assess its diagnostic value of breast cancer.
Objectives
This study is based on chip technology combined with high-performance liquid chromatography suspended from the body's metabolic pathways of breast cancer patients to find the dynamic tumor markers to explore the intrinsic correlation between them, to analyze HIF-1α, Her-2ECD, glycine and alanine acid changes and clinical pathological factors and prognosis and study the prognosis of breast cancer. And to further explore the value and significance of her-2ECD, HIF-la, Ala and Gly in the diagnosis of breast cancer.
Methods
1. Cases from Breast surgery of Ningbo University School of Medicine Affiliated Hospital from 2006 to 2008, Primary breast infiltrating ductal carcinoma:117 cases. Postoperative recurrence of breast infiltrating ductal carcinoma:10 cases. Benign breast tumor:20 cases. breast cosmetic surgery of the health:31 cases(The normal control group). Primary invasive ductal breast cancer were not treated by preoperative radiotherapy, chemotherapy, endocrine therapy and immunotherapy.
2. Serum specimen collection:with the patient's own consent, each selected object is collected blood 10ml in median cubital vein in the early morning. Put them aside for 120 minutes, then take serum after 3000r/min decentralization for 20 minutes, number, and preserve in -80℃refrigerator, screening serum of the breast invasive ductal carcinoma and benign tumor and breast cosmetic surgery of the health patients for inspection according to surgery pathological findings.
3. The collection of tissue samples:with the consent of selected cancer patients, in which surgery would be surgical resection of malignant or benign tumor specimens were cut without necrosis of tumor tissue, each about 1g, and the removal of breast cosmetic surgery at the same time normal tissue of about 1g, and then rinse off bloody with ice saline, immediately placed in liquid nitrogen and stored at -80℃low temperature refrigerators. Before test, take some tumor tissues, respectively, and uonicate to 10% homogenatation under the ice bath after weighing with the ice-cold 0.9% saline by the ratio of 150mg:1.5ml,4000r/min (centrifugal radius 8cm) centrifuge for 10min.The supernatant stored frozen for testing.
4. Sample detection:Serum HIF-1αand Her-2ECD content by suspension chip detection kit (detection range>1pg), and enzyme-linked immunosorbent assay (ELISA) is determinated as controls (HIF-la detection range:10000-156pg; Her-2ECD detection range:> 0.39 ng/ml). Serum and tissues of glycine and alanine are tested by high-performance liquid chromatography, using software packages to calculate concentration.
5. Statistical analysis:SPSS13.0 data were used, all data are mean±standard deviation (x±s). Use analysis of variance (F test)to compare between the two groups. T test was used to compare among two groups, P<0.05 for the difference, there were statistical significances. On the liquid-phase ELISA or HPLC-chip method and the results of tests were using Spearson rank correlation analysis. Test level,α=0.05.
Results
1. Clinical Case Data:178 cases in this group were female, age distribution from 27 to 91 years old, with a median age of 49.2 years old. Primary invasive ductal breast carcinoma of 117 patients, aged from 27 to 91 years old (mean 51.56 years); postoperative recurrence of breast invasive ductal carcinoma of 10 cases, from 35 to 57 years old (mean 47.6 years); 20 cases of benign breast tumor, age 40 to 63 years old (mean 50.38 years); breast cosmetic surgery of 31 cases(The normal control group), aged from 27 to 65 years old (mean 48.77 years); Four groups selected for the patient's age were not statistically significant (P>0.05), All women.Primary invasive ductal breast cancer were not associated preoperative radiotherapy, chemotherapy, endocrine therapy and immunotherapy, clinical pathological stage:Ⅰperiod of 25 cases (21.5%),Ⅱperiod of 61 cases (52.1%),Ⅲperiod of 19 cases (16.2%);Ⅳ,12 cases (10.3%) histopathological grading:G1 phase 12 cases (10.3%), G2 phase 41 cases (35.0%), G3 period of 64 cases (54.7%); tumor size: T1 group≤2cm 41 Li (35.0%), T2 Group> 2cm,≤5cm 61 Li (52.1%), T3 group> 5cm15 cases (12.8%); lymph node metastasis:69 cases without metastasis (59.0%), the transfer of 48 patients (41%).
2. Results of suspension micro-array test
2.1 Primary invasive ductal carcinoma (166.18±59.15) and recurrent invasive ductal carcinoma (231.72±38.39) of serum levels of HIF-1αwere higher than benign tumor group (47.77±15.26) and normal control group (47.84±11.36) (P<0.01); Recurrent infiltrating ductal carcinoma of HIF-1αserum levels were significantly higher than those in primary invasive ductal carcinoma of (P<0.01和P<0.05); There were not statistically significant between the benign tumor group and normal control group serum and tissue HIF-1αlevels. Primary invasive ductal carcinoma of serum levels of HIF-1 levels in clinical and pathological staging of one (150.96±38.85), between the two groups (149.52±52.96) there were no statistical significance(P>0.05), but there were a significant difference between three (189.03±60.36), four stages (249.54±44.84) and one, two stages (P<0.01); and with reduce histological grade (well-differentiated, moderately differentiated and poorly differentiated), its content gradually elevated (119.80±23.28 154.39±52.67,182.42±61.79), there were a significant difference (P<0.01); tumor when the diameter of T3 (diameter>5cm), the serum levels of HIF-1 were significantly higher (215.19±57.11) than the other two groups (T1,T2) (152.42±41.05,161.82±61.69), there were a significant difference (P<0.01); Tumor diameter, T1, T2 were not statistically significant (P>0.05). Lymph node metastasis in patients with serum levels of HIF-1 were significantly higher (197.48±55.28) than that in patients with lymph node metastasis did not occur (144.40±51.80), there were a significant difference(P<0.01), progesterone receptor-negative expression (186.16±59.35) was significantly higher than positive expression(153.23±55.69), there were a significant difference (P<0.01); expression of estrogen receptor-positive (159.91±56.41) and negative expression (179.20±63.27) had no significant difference (P>0.05).
2.2 Primary invasive ductal carcinoma (11.20±6.116) and invasive ductal carcinoma of recurrence (17.61±3.63) of serum Her-2ECD levels were significantly higher than benign (4.91±3.40) and normal control group (4.56±2.95) (P<0.01), Recurrent infiltrating ductal carcinoma higher than primary invasive ductal carcinoma (P<0.01); There were no statistical significance between the benign tumor group and normal control serum Her-2ECD content(P>0.05).Primary invasive ductal breast cancer patients with Her-2ECD level of content and serum levels of HIF-1 were similar, to the clinical pathological staging one(7.89±2.94), two (9.30±4.48), there were no statistically significant (P>0.05), but there is a significant difference between three (15.44±6.38), four stages (21.67±2.76) and one, two stages (P<0.01); tumor tissue with low (13.04±6.53) credit-based were higher than those higher degree of differentiation (in the differentiation and well-differentiated) (9.33±5.33,7.80±1.12), there were a significant difference (P<0.01); Her-2ECD level of concentration with the increase of tumor diameter for increased levels (T3>T2>T1) (18.02±5.57,11.17±6.03,7.98±3.35), there is a significant difference (P<0.01); Lymph node metastasis (15.75±7.06) than those without metastasis (8.04±2.15) in patients with serum Her-2ECD levels were significantly increased, there were a significant difference (P<0.01); Estrogen and progesterone receptor positive and negative expression of Her-2ECD between the serum levels were different, and negative expression was significantly higher (13.49±6.43,13.05±6.42) than positive expression (10.10±5.68,10.01±5.64), there is a significant difference (P<0.01).
3. Results of high-performance liquid chromatography
3.1 Analysis found that serum Ala (338.57±97.37), Gly (176.06±46.9) content in the primary, recurrent invasive ductal carcinoma (240.71±74.33,135.49±31.34) were lower than the normal benign tumor group (414.49±64.94,320.22±42.84) and control group (423.37±80.57,342.19±48.15) (P<0.01); That tissues Ala (177.14±67.99), Gly (147.92±56.74) content in the primary, recurrent invasive ductal carcinoma (224.62±44.96,204.64±40.94) were lower than the normal benign tumor group (437.44±58.10,327.32±46.93) and control group too (406.50±66.85,338.06±49.60) (P<0.01);Ala, Gly in recurrent infiltrating ductal carcinoma group were significantly higher than those in primary invasive ductal carcinoma (P<0.01和P<0.05); While the Ala, Gly in the serum of recurrent infiltrating ductal carcinoma were significantly lower than that of primary invasive ductal carcinoma (P<0.01和P<0.05); Ala, Gly levels in serum and tissues were no statistical significance between the benign tumor group and normal control group.
3.2 Primary invasive ductal carcinoma of the level of serum Ala levels reduce with the clinical and pathological stage gradually increase (433.51±67.69, 342.90±74.78,272.42±60.76,210.05±82.32), there were a significant difference between the levels of different stages (P<0.01); With the histological grade (pathology classification) increased, the level of serum Ala levels reduce (389.91±76.27,362.99±92.43,313.29±97.36), there were a significant difference between different groups (P<0.01); Serum Ala levels and tumor volume is also closely related to tumor volume increased with decreasing concentration(421.19±75.45,323.10±87.08,241.02±43.87), there are significant differences. And lymph node metastases were also inter-related occurred in patients, with lymph node metastasis (297.72±96.01) in Ala serum levels were significantly lower than the transfer did not occur (366.98±88.35) in patients with the level, there were a significant difference (P<0.01); Estrogen (343.37±90.31,328.58±111.27) and progesterone (349.52±96.23 321.67±97.75) receptors and serum in Ala levels were not statistically significant (P>0.05).
3.3 As to primary invasive ductal carcinoma of Gly serum levels in clinical and pathological staging, there were no significant difference betweenⅠ(202.88±37.91),Ⅱperiod (186.18±36.57) (P>0.05), there were a significant difference betweenⅠ,Ⅱperiod andⅢ(152.54±30.75),Ⅳperiod (101.78±45.21) (P<0.01), its content reduced gradually with the classification increased; In tumor cell differentiation, there were no statistical significance between the well-differentiated group (217.50±25.89) and moderately differentiation group (199.12±39.35) (P>0.05), there were statistically significant between the well-differentiated group, moderately differentiation group and poorly differentiation group (153.520±42.089) (P<0.01), with the reduction in the degree of differentiation Gly levels were gradually reduced; Gly serum levels and tumor volume were also closely related to tumor volume increased with decreasing concentration (195.832±37.076, 177.103±45.486,130.260±43.177), there are significant differences(P<0.01). And lymph node metastases were also inter-related occurred in patients with lymph node metastasis, in the Gly serum levels (152.158±46.436) were significantly lower than the transfer did not occur in patients with the level (192.69±39.85), there were a significant difference (P<0.01); Estrogen (181.75±42.12,164.22±54.41) and progesterone receptors (182.73±45.163, 165.767±48.31) and serum of Gly levels were not statistically significant (P> 0.05).
4. Correlation between HIF-1α, Her-2ECD and Ala, Gly:Spearson rank correlation analysis showed that HIF-1αwere positive correlation with Her-2ECD in serum (r=0.533,p=0.000);Ala, (r=-0.429, p=0.000) Gly (r=-0.481, p=0.000) content was negatively correlated with changes in breast cancer serum and tissue. It was closely negatively correlated between HIF-1α, Her-2ECD and Ala (r=-0.403, r=-0.413, p=0.000), Gly (r=-0.509, r=-0.481, p=0.000) in breast invasive ductal carcinoma serum, and showed a closely positive correlation with Ala (r=0.973, r=0.969, p=0.000), Gly (r=0.534, r=0.533, p=0.000) in organization. There were no statistically significance between the normal control group and benign tumors(P>0.05).
5. HIF-1α(87.1%) and Gly(96.6%) is higher than Her-2CED(69.7%) and Ala(67.4%). in diagnosis on breast cancer. Early diagnosis on breast of HIF-1αis higher than Her-2CED(P<0.01); Her-2CED(84.6%) is higher than HIF-1α(67.5%), Ala(64.1%) and Gly(65.8%) in lymph node metastasis of breast cancer. Her-2CED is superior to HIF-1α(P<0.01) in lymph node metastasis of breast cancer.
6. Through the correlation analysis, discriminant analysis and ROC curve analysis, suspended chip technology the ability to detect a single target goal of not less than ELISA kit, test results and contrast the results of tests kits was no significant difference, especially for the determination of the value of tumor samples, suspended Chip kit and the control kit relatively good correlation (r=0.995, r= 0.998, p=0.000)。
Conclusion
1. In serum, HIF-1αand Her-2ECD can be detected with high sensitivity, high-throughput and reliability by using Suspension Array Technology which had a better auxiliary value than ELISA kit in the diagnosis of breast cancer. Therefore, this theme had a viable method and a reliable result, that can be used for research project.
2. The lever of HIF-1αand Her-2ECD in serum of breast invasive ductal carcinoma were overexpression, it were an indication of poor prognosis of the patients, its overexpression indicates that it played an important role in tumorigenesis and tumor induction. So dynamic detection of HIF-1αand Her-2ECD can be used as a real-time, dynamic information, to provide assistance for the development of making treatment program. Therefore, it was important for us to detect the level of HIF-1αand Her-2ECD in serum in the recurrence, early assessment of metastasis and prognosis of breast invasive ductal carcinoma.
3. Ala and Gly involved in anabolic and energy metabolism of the tumor cells of the breast invasive ductal carcinoma, played an important role in the occurrence, development and progression of the breast invasive ductal carcinoma, and may become a new target of the targeted therapy.
4. In serum, HIF-1α、Her-2ECD、Ala and Gly can be used as the early diagnosis of breast cancer, some more comprehensive indicators to evaluate the degree of malignancy, can reflect the biological characteristics of the breast invasive ductal carcinoma. With the basic and clinical research of HIF-1α、Her-2ECD、Ala and Gly in serum going further, it could be one of means in cancer treatment and prevention for targeted treatment.
引文
1.徐光炜.谈乳腺癌的普查.中国医药导报.2010；2(7)：1-3
2. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents:defining priorities to reduce cancer dispariti-es in different geographic regions of the world. J Clin Oncol,2006,24(14):2137-2150.
3.张思维,陈万青,孔灵芝,等.中国部分市县1998-2002年恶性肿瘤的发病与死亡.中国肿瘤,2006,15(7)：430-448
4.陈可欣,何敏,董淑芬,等.天津市女性乳腺癌发病率死亡率和生存率分析.中华肿瘤杂志,2002,6(24)：573-575
5. Ranson M, Tian Z, Andronicos NM, et al. In vitro cytotoxicity of bismuth-213 (213Bi)-labeled-plasminogen activator inhibitor type 2 (alpha-PAI-2) on human breast cancer cells. Breast Cancer Res Treat.2002;71(2):149-59.
6.吴敏,张建新,庞利群.乳腺癌微转移的研究进展.江苏大学学报(医学版),2007；4(17)：366-367
7.王耕,乳腺癌组织中缺氧诱导因子-1α的表达.肿瘤防治研究.2006,6(33)：391-393
8. John R,Griffiths, Marion Stubbs Opportunities for studying cancer by metabolom-ics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Advan. Enzyme Regul.2003 (43):67-76
9. EH Gortl, AJ Groot 1, TLP Derks vande Venl,et al, Hypoxia-inducible f actor-1 a expression requires PI 3-kinase activity and correlates with Aktl phosphorylation in invasive breast carcinomas. Oncogene,2006 (25),6123-6127
10. Robey IF, Lien AD, Welsh SJ,et al, Hypoxia-Inducible Factor-la and the Glycolytic Phenotype in Tumors 1, Neoplasia.2005,4(7):324-330
11.马超,周庚寅,肖颖,等.HIF-1αP-gp在腋淋巴结阴性乳腺癌组织中的表 达及意义.中国肿瘤临床,2007：1(34)：10-13
12.孙媛媛,徐向英.缺氧诱导因子-1α和血管内皮生长因子在乳腺癌中的表达及相关性.实用肿瘤学杂志,2006：4(20)：282-282
1. T.Thorsen, S.J.Maerkl, S.R. Quake, Miero-fluidielarge-sealeintegration.Seienee, 2002,298:580-584.
2. Webb R H.Confoeal optieal mieroseopy. Rep. Prog. Phys.1996,59(3):427-471.
3. J.W.Grate, C.J.B.Lea, A.E.Jarrell, et al. Automated sample Preparation method for suspension arrays using renew able surfaeese Parations with multiplexed flow cytometry fluorescence detection. Analytical Chimica Acta.2003,478:85-98.
4. Hou J. Fluorescent microsPheres and their applications.The Latex Course.2000, 5:17-19.
5. R.Jerrold Fulton, Ralph L. McDad, Perry L.Smith, et al..Advanced multiplexed Analysis with theFloMetrix system.Clinieal Chemstry,1997,43:1749-1756.
6.Watanabe N, Miyamoto M, TokudaY, et al.Serum cerbB-2 in breast cancer patients. Acta Oncol,1994,33(8):901-904.
7. Slamon D J, Clark G M, Wong S G, et al.Human breast cancer:correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science, 1987,235(4785):177-182.
8. Quaranta M, Daniele A, Coviello M, et al. CerbB-2 protein level in tissue and sera of breast cancer patients:a possibly useful clinical correlation.Tumori,2006, 92(4):311-317.
9. Oakman C, Moretti E, Galardi F, et al, The role of topoisomerase Ⅱalpha and HER-2 in predicting sensitivity to anthracyclines in breast cancer patients.Cancer Treat Rev. Cancer Treat Rev.2009,35(8):662-667.
10. Fehm T,Gebauer G,JagerW. Clinical utility of serial serum c-erbB-2 determinations in the follow-up of breast cancer patients. Breast Cancer Res Treat,2002,75 (2):97-106.
11. Imoto S, Kitoh T, Hasebe T, Serum c-erbB-2 levels in monitoring of operable breast cancer patients.Jpn J Clin Oncol,1999,29 (7):336-339.
12. Fornier M, Risio M, Vanpoznak C, et al. HER2 testing and correlation with efficacy of trastuzumab therapy. Oncology (Huntingt),2002,16 (10):1340-1352.
13. Isola J J, Holli K, Oksa H, et al. Elevated erbB-2 oncoprotein levels in preoperative and follow-up serum samples define an aggressive disease course in patients with breast cancer. Cancer,1994,73(3):652-658.
14. Cook G B, Neaman I E, Goldblatt J L, et al.Clinical utility of serum HER-2/neu testing on theBayer Immuno 1 automated system in breast cancer.Anticancer Res, 001,21(2B):1465-1470.
15.Zhang Q,Tang Q,Lu Q, et al, Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia-and serum-induced HIF-1αlpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells. Mol Cancer Ther. 2006;5(5):1227-38.
16.Yamamoto Y,Ibsuki M, Okumura Y, et al. Hypoxia-inducible factor 1alpha is closely linked to an aggressive phenotype in breast cancer. Breast Cancer Res Treat.2008 Aug;110(3):465-475.
17. Bluff JE, Menakuru SR, Cross SS, et al, Angiogenesis is associated with the onset of hyperplasia in human ductal breast disease. Br J Cancer.2009 Aug 18;101(4):666-72.
18.Schindl M, Schoppmann SF, Samonigg H, et al;Austrian Breast and Colorectal Cancer Study Group.Clin Cancer Res. Overexpression of hypoxia-inducible factor-1alpha is associated with an unfavorable prognosis in lymph node-positive breast cancer.2002 Jun;8(6):1831-7.
19.Schindl M, Schoppmann SF, Samonigg H, et al. Overexp ression of hypoxia-inducible factor 1alpha is associated with an unfavorable prognose in lymph node-positive breast cancer. ClinCancer Res,2002,8 (6):1831-1837.
20.Bos R, vander Groep P, Greijer AE, et al. Levels of hypoxia-inducible factor-alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer,2003,97(6):1573-1581.
21. Chen HH,Su WC, Hypoxia-inducible factor-1 alpha correlates with MET and metastasis in node-negative breast cancer.Breast Cancer Res Treat.2007 Jun;103(2):167-75.
22. Bottaro DP, Liotta LA. Out of air is not out of action. Nature,2003,423 (6940): 593-595.
23. Bernards R. Cancer:cues for migration. N ature,2003,425(6955):247-248.
24. Kim KK, Lee JJ, Yang Y, et al. Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells.Carcinogenesis.2008;29(4):704-12. Epub 2008 Feb 6.
25.Bartlett JM, Munro A, Cameron DA, et al. Type 1 receptor tyrosine kinase profiles identify patients with enhanced benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial. J Clin Oncol.2008 N;26(31):5027-35.
26. Ma C, Zhou GY, Xiao Y, et al. Reversion the multidrug resistance of human breast carcinoma cells by RNA interference targeting HIF-1 alpha gene. Zhonghua Bing Li Xue Za Zhi.2006;35(6):357-60.
27.柳光宇,沈坤炜,邵志敏,等.乳腺癌组织缺氧与雌激素受体-α的关系.中国癌症杂志,2004,14(2)：127-130.
28.Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexp ressing cells. Cancer Res,2005,65 (8):3257-3263.
29. Giatromanolaki A, Koukourakis MI, Simopoulos C, et al, Cancer Biol Ther. Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original c-erbB-2/HIF1alpha phenotype,2007;6(2):153-5.
30. Benezra R, Henke E, Ciarrocchi A, et al, Induction of complete regressions of oncogene-induced breast tumors in mice. Cold Spring Harb Symp Quant Biol. 2005;70:375-81.
31. Li YM, Zhou BP, Deng J, et al, A hypoxia-independent6202hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells.Cancer Res.2005,15;65(8):3257-63.
32. Giatromanolaki A, Koukourakis MI, Simopoulos C, et al. c-erbB-2 related aggressiveness in breast cancer is hypoxia inducible factor-1alpha dependent.Clin Cancer Res.2004;10(23):7972-7.
33. Menendez JA, Vellon L, Oza BP, et al. Does endogenous fatty acid metabolism allow cancer cells to sense hypoxia and mediate hypoxic vasodilatation? Characterization of a novel molecular connection between fatty acid synthase (FAS) and hypoxia-inducible factor-1alpha (HIF-1alpha)-related expression of vascular endothelial growth factor (VEGF) in cancer cells overexpressing her-2/neu oncogene.J Cell Biochem.2005;94(5):857-63.
34. Howard EM, Lau SK, Lyles RH, et al, Expression of e-cadherin in high-risk breast cancer. J Cancer Res Clin Oncol.2005;131(1):14-8.
35. Laughner E, Taghavi P, Chiles K, et al, Mol Cell Biol. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.2001;21(12):3995-4004.
36. Matsumoto K, Arao T, Tanaka K, et al. mTOR signal and hypoxia-inducible factor-1 alpha regulate CD133 expression in8-Azaguaninecancer cells.Cancer Res.2009;69(18):7160-4.
37. Liu M, HowesA, Lesperance J, Antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer. Cancer Res,2005,65 (12):5325-5336
38. Shafee N, Kaluz S, Ru N, et al, PI3K/Akt activity has variable cell-specific effects on expression of HIF target genes, CA9 and VEGF, in human cancer cell lines.Cancer Lett.2009;282(1):109-15.
1. Taylor J, King R D, Fiehn O, et al. App lication ofmetabolomics to plant genotype discrimination using statistics and machine learning. Bioinform atics,2002,18 (10):241-248.
2. Reo N V. NMR2based metabolomics. D rug Chem Toxicol,2002,25 (4):375-382.
3.桑志红,杨松成.液相色谱-质谱联用技术及其在药物分析中的应用.解放军药学学报,1999,15(2)：28-31.
4:Miyoshi Y, Hamase K, Tojo Y, et al, Determination of D-serine and D-alanine in the tissues and physiological fluids of mice with various D-amino-acid oxidase activities using two-dimensional high-performance liquid chromatography with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877(24):2506-12.
5:Penteado JC, Rigobello-Masini M, Liria CW, et al, Fluorimetric determination of intra-and extracellular free amino acids in the microalgae Tetraselmis gracilis (Prasinophyceae) using monolithic column in reversed phase mode.J Sep Sci. 2009;32(15-16):2827-34.
6:Bhushan R, Kumar R. Analysis of multicomponent mixture and simultaneous enantioresolution of proteinogenic and non-proteinogenic amino acids by reversed-phase high-performance liquid chromatography using chiral variants of Sanger's reagent.Anal Bioanal Chem.2009;394(6):1697-705.
7:Giuntini F, Bourre L, Macrobert AJ, et al. Quantitative determination of 5-aminolaevulinic acid and its esters in cell lysates by HPLC-fluorescence.J Chromatogr B Analyt Technol Biomed Life Sci.2008;875(2):562-6.
8. Rahman MM, Takafuji M, Ihara H. Synthesis and assessment of molecular recognizability by RP-HPLC of an N-alkyl-beta-Ala-L-Phe-derived organic phase with self-assembling ability.Anal Bioanal Chem.2008;392(6):1197-208.
9. Bergero D, Assenza A, Schiavone A, et al. Amino acid concentrations in blood serum of horses performing long lasting low-intensity exercise. J Anim Physiol Anim Nutr (Berl).2005;89(3-6):146-50.
10. Krstanovic M, Brgles M, Halassy B, et al. Purification and characterization of 1,(l/d)-aminopeptidase from Guinea pig serum. Prep Biochem Biotechnol. 2006;36(2):175-95.
11. Hanko VP, Rohrer JS. Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection. Anal Biochem.2004;324(1):29-38.
12. Josemans AI, Zweygarth E. Amino acid content of cell cultures infected with Cowdria ruminantium propagated in a protein-free medium. Ann N Y Acad Sci. 2002;969:141-6.
13. Morikawa A, Hamase K, Inoue T, et al. Determination of free D-aspartic acid, D-serine and D-alanine in the brain of mutant mice lacking D-amino acid oxidase activity. J Chromatogr B Biomed Sci Appl.2001;757(1):119-25.
14.郑璐侠,邵泓,陈钢,等,13种氨基酸和牛磺酸的柱前衍生化HPLC测定.中国医药工业杂志,2008,39(8)：610-612。
15.Yang J,Xu G W,Kong H W,et al.Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer..J Chrom atogr,2002,780(1):27-33.
16.Zheng Y F,Xu G W,L iu D Y,et al..Study of urinary nucleosides as biological marker in cancer patients analyzed by micellar electrokinetic capillary chromatography.E ctrophoresis,2002,23(24):4104-4109.
17..Friedecky D,Adam T,Bartak P. Cap illary electrophoresis for detection of inherited disorders of purine and pyrim idine metabolism:A selective app roach.E lectrophoresis,2002,23(4):565-571.
18.Claudia C, BirarelliM, NolaM, et al. Automated high-perform-ance liquid chromatographic method for the determination of ho-mocysteine in p lasma samp les.J Chrom atogrA,1999,846 (122):93-100.
19. Ben-yoseeph O, Badar-Goffer R S, Morris PG, et al. Biochem J,1993,291 (Pt 3):915-919.
20.John R. Griffiths, Marion Stubbs Opportunities for studying cancer by metabolom-ics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Advan. Enzyme Regul.2003 (43):67-76
21.Griffiths J R, Stubbs M, Opportunities for studying cancer by metabolomics: preliminary observations on tumors deficient in hypoxia-inducible factor 1. Adv Enzyme Regul,2003,43:67-76.
22. Reo N V. NMR-based metabolomics. Drug Chem Toxicol,2002,25 (4):375-382.
23.Cascino A, Muscaritoli M, Cangiano C,et al.Plasma aminoacid imbalance in patients with lung and breast cancer. Anticancer Res.1995;15(2):507-510.
24. Davies NP, Wilson M, Natarajan K, et al. Non-invasive detection of glycine as a biomarker of malignancy in childhood brain tumours using in-vivo 1H MRS at 1.5 Tesla confirmed by ex-vivo high-resolution magic-angle spinning NMR. NMR Biomed.2010;23(1):80-7.
25. Choi BD, Archer GE, Mitchell DA, et al. EGFRvⅢ-targeted vaccination therapy of malignant glioma.Brain Pathol.2009 Oct;19(4):713-23.
26. LIUHong-Yan, LUGuo-Wei, WANGWei-Zhong. Changes in glycine content in mouse brain during hypoxic preconditioning.Acta Physiologica Sinica. 2001,53(6),461-464。
27. Lu J, Chen ZW. Isolation, characterization and anti-cancer activity of SK84, a novel glycine-rich antimicrobial peptide from Drosophila virilis. Peptides. 2010;31(1):44-50
28. Pfeiffer F, Betz H. Solubilisation of the glycine receptor from the rat spinal cord. Brain Res,1981,226:273-279.
29.Iimuro Y, Bradford BU, Yamashina S, et al. The glutathione precursor L-2-oxothiazolidine-4-carboxylic acid protects against liver injury due to chronic enteral ethanol exposure in the rat. Hepatology,2000,31:391-398.
30.Li X, Bradford BU, Wheeler MD, et al. Dietary glycine prevents peptidoglycan polysaccharide-induced reactive arthritis in the rat:role for glycine-gated chloride channel. Infect Immun,2001,69:5883-5891.
31.Wheeler MD, Rose ML, Yamashima S, et al. Dietary glycine blunts lung inflammatory cell influx following acute endotoxin. Am J Physiol Lung Cell Mol Physiol,2000,279:390-398.
32.Wheeler MD, Stachlewitz RF, Yamashina S, et al. Glycine!gated chloride channels in neutrophils attenuate calcium influx and superoxide production. FASEB J,2000,14:476-484.
33. Stachlewitz RF, Li X, Smith S, et al. Glycine inhibits growth of Tlymphocytes by an IL-2-independent mechanism. J Immunol,2000,164:176-182.
34. Serganov A, Patel DJ. Amino acid recognition and gene regulation by riboswit-ches. Biochim Biophys Acta. Biochim Biophys Acta.2009;1789(9-10):592-611
35.Wang CL, Teo KY, Han B. An amino acidic adjuvant to augment cryoinjury of MCF-7 breast cancer cells.Cryobiology.2008;57(1):52-9.
36.Richardson AD, Yang C, Osterman A, et al. Central carbon metabolism in the progression of mammary carcinoma.Breast Cancer Res Treat.2008;110(2):297-307.
37.Hensley K, Robinson KA, Gabbita SP, et al. Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med,2000,28:1456-1462.
38.Wang HD, Lu XX, Lu DX, et al. Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFalpha production in cardiomyocytes by activating a glycine receptor. Acta Pharmacol Sin.2009;30(8):1107-14.
39. Nakamura Y, Matsumoto S, Mochida T, et al. Glycine regulates proliferation and differentiation of salivary-gland-derived progenitor cells.Cell Tissue Res. 2009;336(2):203-12.
40.王亮,李保义,李庆瑞,胃癌组织氨基酸与肿瘤体积相关性研究。肠外与肠内营养,2000,(17)：41-44.
41.Yuan G, Nanduri J, Bhasker CR, et al. Ca2+/calmodulin kinase dependent activation of hypoxia inducible factor 1 transcriptional activity in cells subjected to intermittent hypoxia. J Biol Chem,2005,280(6):4321-4328.
42.Yuan G, Nanduri J, Khan S, et al. Induction of HIF-1alpha expression by intermittent hypoxia:involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR.J Cell Physiol.2008;217(3):674-85.
43. Lee SH, Heo JS, Han HJ. Effect of hypoxia on 2-deoxyglucose uptake and cell cycle regulatory protein expression of mouse embryonic stem cells:involvement of Ca2+/PKC, MAPKs and HIF-1alpha.Cell Physiol Biochem.2007;19(5-6):269-282.
44.Shen WG, Peng WX, Shao Y, et al. Localization and activity of calmodulin is involved in cell-cell adhesion of tumor cells and endothelial cells in response to hypoxic stress.Cell Biol Toxicol.2007;23(5):323-35.
45.Osada-Oka M, Takahashi M, Akiba S, et al, Eur J Pharmacol. Involvement of Ca2+-independent phospholipase A2 in the translocation of hypoxia-inducible factor-1 alpha to the nucleus under hypoxic conditions.2006;549(1-3):58-62.
46.Yuan G, Nanduri J, Bhasker CR, et al. Ca2+/calmodulin kinase-dependent activation of hypoxia inducible factor 30,6-Djhydroxycholanic Acidtranscription -al activeity in cells subjected to intermittent hypoxia. J Biol Chem.2005;280(6): 4321-4328.
47.Wollheim CB. Beta-cell mitochondria in the regulation of insulin secretion:a new culprit in type 2 diabetes. Diabetologia,2000,43:265-277.
48.Civekek VN, Deeney JT, Shalosky NJ,et al. Regulation of pancreatic β-cell mitochondrial metabolism:influence of Ca2+,substrate and ADP. Biochem J 1996, 318:615-621.
49.Comerford KM, Wallace TJ, Karhausen J, et al. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance(MDR1) gene.Cancer Res,2002,62(12):3387-94.
1. Jemal A, Siegel R, Ward E, et al. Cancer statistics,2007. CA Cancer J Clin,2007, 57(1):43-66.
2. Scheurle D,Jahanzeb M,Aronsohn RS,et al. HER-2/neu expression in archival non-small lung carcinomas using FDA-approved Hercerptin lest. Anticancer Res,2000,20(3B):2091.
3.周爱萍,乳腺癌靶向治疗:Herceptin的研究进展.国外医学肿瘤学分册,2000,8(4)：167.
4.Lufmer D,luke C,Possinger K, serum HER-2/neu in the management of breast cancer patients. Clinic Biochem,2003,36:233.
5.Griffiths J R, Stubbs M, Opportunities for studying cancer by metabolomics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Adv Enzyme Regul,2003,43: 67-76.
6.代志军,高洁,王西京,等.乳腺导管增生癌变过程中HIF-1α的表达变化及意义.中国肿瘤临床与康复,2007,14(1)：20-22.
7.Bos R, Zhong H, Hanmdan FC, et al. Levels of hypoxia inducible factor 1 alpha during breast carcinogenesis. Nat Cancer Inst Monogr,2001,93:309-314.
8.Chen HH, Su WC, Hypoxia-inducible factor-1αlpha correlates with MET and metastasis in node-negative breast cancer.Breast Cancer Res Treat.2007;103(2): 167-75.
9.王耕,乳腺癌组织中缺氧诱导因子-1α的表达.肿瘤防治研究2006,6(33)：391-393.
10. Hao Ls, Zhu X, Qian K, et al. Expression of hypoxia-inducible factor 1 alpha in human breast carclnonla. West China Medical,2006,21(3):445-446.
11. Bos R, Zhong H, Hanmdan FC, et al. Levels of hypoxia inducible factor 1 alpha during breast carcinogenesis. Nat Cancer Inst Monogr,2001,93:309-314.
12. Yang ZF, Poon RT, To J, et al. The potential mh of hypoxia-inducible factor 1 alpha in tumor progression after hypoxia and chemotherapy in hepatocelular carcinoma. Cancer Res,2004,64(15):5496-5503
13.Griffiths J R, Stubbs M, Opportunities for studying cancer by metabolomics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Adv Enzyme Regul,2003,43: 67-76.
14.Edelstein CL, Ling H, Gengaro PE, et al. Effeet of glycine on Prelethal and Postlethal increases in calpain activity in rat renal Proximal tubules. Kidney international,1997,52(5):1271-1278.
15.Wheeler M, Stachlewitz RF, Yamashina S, et al. Glycine-gated chloride channels in neutronphils attenuate calcium influx and superoxide Production. FASEB J,2000,14(3):476-484.
16.Yamashina S, Konno A, Wheeler MD, et al. Endothelial Cells contain a glycine-gated Chloride channel. Nutr Cancer,2001,40(2):197-204.
17.Griffiths J R, Stubbs M, Opportunities for studying cancer by metabolomics: preliminary observations on tumors deficient in hypoxia-inducible factor 1. Adv Enzyme Regul,2003,43:67-76.
18.Yuan G, Nanduri J, Bhasker CR, et al. Ca2+/calmodulin kinase dependent activation of hypoxia inducible factor 1 transcriptional activity in cells subjected to intermittent hypoxia. J Biol Chem,2005,280(6):4321-4328.
19.Edelstein CL, Ling H, Gengaro PE, et al. Effeet of glycine on Prelethal and Postlethal increases in calpain activity in rat renal Proximal tubules. Kidney international,1997,52(5):1271-1278.
20.Koukourakis M I, Simopoulos C, Polychronidis A, et al, The effect of trastuzumab/docatexel combination on breast cancer angiogenesis:dichotomus effect predictable by the HIFI alpha/VEGF pre-treatment status? Anticancer Res. 2003;23(2C):1673-80.
21.Bos R, vander Groep P, Greijer A E, et al. Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer.2003;97(6):1573-81.
22.Yen L, Benlimame N, Nie ZR, et al. Differential regulation of tumor angiogenesis by distinct ErbB homo and heterodimers. Mol Biol Cell. 2002;13(11):4029-44
23. Laughner E, Taghavi P, Chiles K, et al, HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1 alpha (HIF-1 alpha) synthesis:novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol. 2001;21(12):3995-4004.
24. Bos R, Zhong H, Hanrahan CF, et al. Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis.J Natl Cancer Inst.2001;93(4):309-14.
25. Giatromanolaki A, Koukourakis MI, Simopoulos C, et al, Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original c-erbB-2/HIF1alpha phenotype. Cancer Biol Ther.2007;6(2):153-5.
26.Petit T, Borel C, Ghnassia J P, et al. Chemotherapy response of breast cancer depe nds on HER-2 status an d an thracycline doseintensity in the neoadjuvant setting. Clin Cancer Res,2001,7(6):1577-1581
27.Laughner E, Taghavi P, Chiles K, et al. HER2 signaling increases the rate of hypexia-inducible-la synthesis:novel mechan ism for HIF-1 mediated vascular endothelial growth factor Expression. Mole Cell Biol,2001,21(12):3995-4004
28.Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidyli-nositol-3 kinase/Akt in HER2 overexpressing cells. Cancer Res,2005,65:3257-3263.
29. Gort EH, Groot AJ, Derks van de Ven TL, et al. Hypoxia-inducible factor-1alpha expression requires PI 3-kinase activity and correlates with Akt1 phosphoryla-tion in invasive breast carcinomas. Oncogene.2006;25(45):6123-6127.
30. Liu M, HowesA, Lesperance J, Antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer. Cancer Res,2005,65 (12):5325-5336
31. Kim KK, Lee JJ, Yang Y, et al. Macrophage inhibitory cytokine-1 activates AKT and ERX-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells. Carcinogenesis.2008;29(4):704-12.
32. Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells.Cancer Res.2005;65(8):3257-63.
33.Kim KK, Lee JJ, Yang Y, et al. Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells.Carcinogenesis.2008;29(4):704-12.
34.Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexp ressing cells. Cancer Res,2005,65 (8):3257-3263.
35. Li YM, Zhou BP, Deng J, et al, A hypoxia-independent6202hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells.Cancer Res.2005;65(8):3257-63.
36. Shafee N, Kaluz S, Ru N, et al, PI3K/Akt activity has variable cell-specific effects on expression of HIF target genes, CA9 and VEGF, in human cancer cell lines.Cancer Lett.2009;282(1):109-15.
37. Liao JY, Li LL, Wei Q, et al, Arch Biochem Biophys. Heregulinbeta activates store-operated Ca2+ channels through c-erbB2 receptor level-dependent pathway in human breast cancer cells.2007;458(2):244-52.
38. Feinmesser RL, Wicks SJ, Taverner CJ, et al, Biol Chem. Ca2+/calmodulin-dependent kinase Ⅱ phosphorylates the epidermal growth factor receptor on multiple sites in the cytoplasmic tail and serine 744 within the kinase domain to regulate signal generation.1999;274(23):16168-73.
39.Civekek VN, Deeney JT, Shalosky NJ, et al. Regulation of pancreatic β-cell mitochondrial metabolism:influence of Ca2+, substrate and ADP. Biochem J 1996,318:615-621.
40.Giatromanolaki A, Koukourakis MI, Simopoulos C, et al, Cancer Biol Ther. Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original c-erbB-2/HIF1alpha phenotype,2007;6(2):153-5.
41. Benezra R, Henke E, Ciarrocchi A, et al, Induction of complete regressions of oncogene-induced breast tumors in mice. Cold Spring Harb Symp Quant Biol. 2005;70:375-81.
42. Giatromanolaki A, Koukourakis MI, Simopoulos C, et al. c-erbB-2 related aggressiveness in breast cancer is hypoxia inducible factor-1alpha dependent. Clin Cancer Res.2004;10(23):7972-7.
43. Menendez JA, Vellon L, Oza BP, et al. Does endogenous fatty acid metabolism allow cancer cells to sense hypoxia and mediate hypoxic vasodilatation? Characterization of a novel molecular connection between fatty acid synthase (FAS) and hypoxia-inducible factor-1alpha (HIF-1alpha)-related expression of vascular endothelial growth factor (VEGF) in cancer cells overexpressing her-2/neu oncogene.J Cell Biochem.2005;94(5):857-63.
44. Howard EM, Lau SK, Lyles RH, et al. Expression of e-cadherin in high-risk breast cancer. J Cancer Res Clin Oncol.2005;131(1):14-8.
45. Laughner E, Taghavi P, Chiles K, et al, Mol Cell Biol. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.2001;21(12):3995-4004.
46. Yuan G, Nanduri J, Khan S, et al. Induction of HIF-1alpha expression by intermittent hypoxia:involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR.J Cell Physiol.2008;217(3):674-85.
47. Tajima N, Schonherr K, Niedling S,et al, Ca2+-activated K+ channels in human melanoma cells are up-regulated by hypoxia involving hypoxia-inducible factor-1alpha and the von Hippel-Lindau protein. J Physiol.2006 1;571(Pt 2):349-59.
1.Nicholls AW, Holmes E, Lindon J C, et al. Metabonomic investigations into hydrazine toxicity in the rat. Chem Res Toxicol.2001,14(8):975-987.
2.Art hur J M, Thongboonker DV, Scherzer JA, et al. Differential expression of proteins in renal cortex and medulla:a proteomic approach. Kidny Int,2002,62(8): 1314-1318
3.Nicholson J K, Lindon J C, Holmes E, et al. Metabonomics:understanding the metabolic responses of living systems to pathophysiological stimuli viamultivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica,1999,29(11):1181-1189
4.Waters NJ, Holmes E, Warterfield CJ, et al, NMR and pattern recognition studies on liver extracts and intact livers from rats treated with anaphthylisonthiocyanate. Biochem Pharmacol,2002,64:67
5.Tavlor J, King RD, Altmann T, et al. Application of metabolomics to plant genotype discrimination using statistics and machine learning. Bioinformatics, 2002,18(Suppl 2):S241-S248
6.朱勇飞,张天宝.代谢组学在毒理学研究中的应用.国外医学卫生学分册,2005,32(3)：156-159
7.Fiehn O. Metabolomics the link between genotypes and phenotypes. Plant Mol Biol,2002,48(1/2):155-171
8. Douglas B K.Metabolom ics and system s biology:making sense of the soup.Cur Opin in M-icrobiology,2004,7:296
9. Greenlee RT, Murray T, Bolden S, et al.Cancer statistics,2000. CA Cancer J Clin. 2000;50(1):7-33
10.Tiwari RC, Ghosh K, Jemal A, et al. A new method of predicting US and state -level cancer mortality counts for the current calendar year. CA Cancer J Clin. 2004;54(1):30-40.
11.Moysich KB, Menezes RJ, Baker JA, et al Environmental exposure to polychlori-nated biphenyls and breast cancer risk. Rev Environ Health.2002;17(4):263-77.
12. Bray GA. The underlying basis for obesity:relationship to cancer. J Nutr. 2002;132(11 Suppl):3451S-3455S.
13.McTiernan A, Rajan KB, Tworoger SS, et al.Adiposity and sex hormones in postmenopausal breast cancer survivors. J Clin Oncol.2003;21(10):1961-6.
14.Key T, Appleby P, Barnes I, et al. Endogenous sex hormones and breast cancer in postmenopausal women:reanalysis of nine prospective studies. J Natl Cancer Inst.2002;94(8):606-16.
15.Klymachyov A, Dalai N.Spinning crystals leads to significant enhancement in 13C spectral resolution in MAS experiments on organic compounds:a new aid in studying phase transitions. Solid State Nucl Magn Reson.1997;9(1):85-9.
16. Castrillo JI, Hayes A, Mohammed S, et al. An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry. Phytochemistry.2003;62(6):929-37.
17. Potts BC, Deese AJ, Stevens GJ,et al.NMR of biofluids and pattern recognition: assessing the impact of NMR parameters on the principal component analysis of urine from rat and mouse. J Pharm Biomed Anal.2001;26(3):463-76.
18. Beckwith-Hall BM, Brindle JT et al.Application of orthogonal signal correction to minimise the effects of physical and biological variation in high resolution 1H NMR spectra of biofluids. Analyst.2002;127(10):1283-8.
19. Daykin CA, Corcoran O, Hansen SH et al.Application of directly coupled HPLC NMR to separation and characterization of lipoproteins from human serum. Anal Chem.2001;73(6):1084-90.
20.Griffin JL, Williams HJ, Sang E,et al.Metabolic profiling of genetic disorders:a multitissue (1)H nuclear magnetic resonance spectroscopic and pattern recogni-tion study into dystrophic tissue. Anal Biochem.2001;293(1):16-21.
21.Brindle JT, Antti H, Holmes E, et al.Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nat Med.2003;9(4):477.
22. Waters NJ, Holmes E, Waterfield CJ, et al.NMR and pattern recognition studies on liver extracts and intact livers from rats treated with alpha-naphthylisothiocya-nate. Biochem Pharmacol.2002;64(1):67-77
23.Griffin J L,Shockcor J P. Metabolic profiles of cancer cells. Nat Rev Cancer.2004 Jul;4(7):551-61
24.Cheng LL, Chang IW, Smith BL, et al.Evaluating human breast ductal carcinomas with high-resolution magic-angle spinning proton magnetic resonance spectroscopy. J Magn Reson.1998;135(1):194-202.
25. Fiehn O. Metabolomics-the link between genotypes and phenotypes. Plant Mol Biol.2002;48(1-2):155-71.
26. Mackinnon WB, Barry PA, Malycha PL, et al.Fine-needle biopsy specimens of benign breast lesions distinguished from invasive cancer ex vivo with proton MR spectroscopy. Radiology.1997;204(3):661-6.
27.许国旺,杨军.色谱,2003,21(4)：316-320.
28.Leach MO, Verrill M, Glaholm J, et al.Measurements of human breast cancer us-ing magnetic resonance spectroscopy:a review of clinical measurements and a report of localized 31P measurements of response to treatment. NMR Biomed. 1998;11(7):314-40.
29. Merchant TE, Kasimos JN, Vroom T, et al.Malignant breast tumor phospholipid profiles using (31)P magnetic resonance. Cancer Lett.2002;176(2):159-67.
30. Barzilai A, Horowitz A, Geier A, et al.Phosphate metabolites and steroid horm-one receptors of benign and malignant breast tumors. A Nuclear Magnetic Reso-nance study. Cancer.1991;67(11):2919-25.
31. Singer S, Souza K, Thilly WG. Pyruvate utilization, phosphocholine and adeno-sine triphosphate (ATP) are markers of human breast tumor progression:a 31P-and 13C-nuclear magnetic resonance (NMR) spectroscopy study. Cancer Res. 1995;55(22):5140-5.
32. Rivenzon-Segal D, Margalit R, Degani H.Glycolysis as a metabolic marker in ort-hotopic breast cancer, monitored by in vivo (13)C MRS. Am J Physiol Endocrinol Metab.2002;283(4):E623-30.
33. Katz-Brull R, Seger D, Rivenson-Segal D, et al.Metabolic markers of breast cancer:enhanced choline metabolism and reduced choline-ether-phospholipid synthesis. Cancer Res.2002;62(7):1966-70.
34. Yu JX, Kodibagkar VD, Liu L, et al.A 19F-NMR approach using reporter mole-cule pairs to assess beta-galactosidase in human xenograft tumors in vivo. NMR Biomed.2008;21(7):704-12.
35. Holmberg L, Jakobsson U, Berglund A, et al.Failure to detect early breast cancer using in vitro nuclear magnetic resonance spectroscopy of plasma. Br J Cancer. 1993;68(2):389-92.
36. Raffelt K, Moka D, Sullentrop F,et al.Systemic alterations in phospholipid con-centrations of blood plasma in patients with thyroid carcinoma:an in-vitro (31)-P high-resolution NMR study. NMR Biomed.2000;13(1):8-13.
37. Boss EA, Moolenaar SH, Massuger LF, et al.High-resolution proton nuclear magnetic resonance spectroscopy of ovarian cyst fluid. NMR Biomed.2000; 13 (5):297-305.
38. H.S.Shaw,M.J.Ellis, Letrozole in the treatment of breast cancer. Expert Opin Pharmacother.2002;3(5):607-17.
39. Glunde K, Ackerstaff E, Natarajan K, et al.Real-time changes in 1H and 31P NMR spectra of malignant human mammary epithelial cells during treatment with the anti-inflammatory agent indomethacin. Magn Reson Med.2002;48(5) :819-25.
40. Lonning PE, Geisler J, Johannessen DC, et al.Pharmacokinetics and metabolism of formestane in breast cancer patients. J Steroid Biochem Mol Biol.2001;77(1): 39-47.
41. Odunsi K, Wollman RM, Ambrosone CB, et al.Detection of epithelial ovarian cancer using 1H-NMR-based metabonomics. Int J Cancer.2005;113(5):782-8.
42. Whitehead TL, Monzavi-Karbassi B, Jousheghany F,et al.1H-NMR metabolic markers of malignancy correlate with spontaneous metastases in a murine mam-mary tumor model. Int J Oncol.2005;27(1):257-63.
43. Stanley EG, Bailey NJ, Bollard ME,et al.Sexual dimorphism in urinary metabo-lite profiles of Han Wistar rats revealed by nuclear-magnetic-resonance-based metabonomics. Anal Biochem.2005;343(2):195-202.
44. Slim RM, Robertson DG, Albassam M,et al. Effect of dexamethasone on the met-abonomics profile associated with phosphodiesterase inhibitor-induced vascular lesions in rats. Toxicol Appl Pharmacol.2002;183(2):108-9.
45. Lenz EM, Bright J, Wilson ID, Hughes A,et al. Metabonomics, dietary influenc-es and cultural differences:a 1H NMR-based study of urine samples obtained from healthy British and Swedish subjects. J Pharm Biomed Anal.2004;36(4): 841-9.
46. Tolstikov VV, Lommen A, Nakanishi K,et al. Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics. Anal Chem.2003 Dec 1;75(23):6737-40.
47. Sugimoto M, Kikuchi S, Arita M,et al. Large-scale prediction of cationic metabo -lite identity and migration time in capillary electrophoresis mass spectrometry using artificial neural networks. Anal Chem.2005;77(1):78-84.
48. Pham-Tuan H, Kaskavelis L, Daykin CA, et al. Method development in high-per-formance liquid chromatography for high-throughput profiling and metabonom-ic studies of biofluid samples. J Chromatogr B Analyt Technol Biomed Life Sci. 2003;789(2):283-301.
49.桑志红,杨松成.液相色谱-质谱联用技术及其在药物分析中的应用.解放军药学学报,1999,15(2)：28-31.
50.Yang J,Xu G W,Kong H W,et a.lA rtificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer..J Chrom atogr,2002,780(1):27-33.
51.Zheng Y F,Xu G W,L iu D Y,et al. Study of urinary nucleosides as biological marker in cancer patients analyzed by micellar electrokinetic capillary chromatography.E ctrophoresis,2002,23(24):4104-4109.
52.Friedecky D,Adam T,Bartak P.Cap illary electrophoresis for detection of inherited disorders of purine and pyrim idine metabolism:A selective app roach.E lectrophoresis,2002,23(4):565-571.
53. Sitter B, Sonnewald U, Spraul M, et al. High-resolution magic angle spinning MRS of breast cancer tissue. NMR Biomed.2002 Aug;15(5):327-37.
54. Plumb RS, Stumpf CL, Gorenstein MV, et al. Metabonomics:the use of electro-spray mass spectrometry coupled to reversed-phase liquid chromatography sho-ws potential for the screening of rat urine in drug development. Rapid Commun Mass Spectrom.2002;16(20):1991-6.
55.Xiang R, Shi Y, Dillon DA, et al.2D LC/MS analysis of membrane proteins from breast cancer cell lines MCF7 and BT474. J Proteome Res.2004;3(6):1278-1283.
56.Zang L,Palmer Toy D,Hancock WS, et al.Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 160/180 isotopic labeling. J Proteome Res.2004;3(3):604-12.
57.Chong BE, Yan F, Lubman DM,et al. Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates:a novel two-dimensional liquid chromatography/mass spectrometry method.Rapid commun Mass spectrum.2001;15(4):291-6.
58.Mbeunkui F,Metge BJ,Shevde LA,et al.Identification of differentially secreted biomarkers using LC-MS/MS in isogenic cell lines representing a progression of breast cancer. J Proteome Res.2007;6(8):2993-3002.
59.Byun JA,Lee SH, Jung BH,et al,Analysis of polyamines as carbamoyl derivatives in urine and serum by liquid chromatography-tandem mass spectrometry.Biomed chromatogr.2008;22(1):73-80
60.Murph M, Tanaka T,Pang J, et al. Liquid chromatography mass spectrometry for quantifying plasma lysophospholipids:potential biomarkers for cancer diagnosis. Methods enzymol.2007;433:1-25.
61.Whiteaker JR,Zhang H,Zhao L,et al.Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer. J Proteome Res.2007;6(10):3962-75.
62. Qin F, Zhao YY, Sawyer MB,et al. Hydrophilic interaction liquid chromatography-tandem mass spectrometry determination of estrogen conjugates in human urine. Anal Chem.2008;80(9):3404-11.
63.沈朋,曾真,程翼宇.乳腺癌血清蛋白质组与尿代谢物组协同分析研究.中华检验医学杂志.2005,28(7)：156-159
64.Shi Y,Xiang R,Horvath C, et al. Quantitative analysis of membrane proteins from breast cancer cell lines BT474 and MCF7 using multistep solid phase mass tagging and 2D LC/MS.J Proteome Res.2005 4(4):1427-33.
1 Semenza GL, ADmi F, Booth G, et al. Structural andfunctionalan alysis of hypoxia-nducible factor 1. Kidney Int,1997;51:553-55
2 Minet E, Michel G, Remacle P, et al. Role of HIF-1 as a transciption factor involved in embryonic development, ca/leer progression and apoptosis. Int J Mol Med,2000;5:253-259
3 Maxwell PH, Dachs GU, Gleadle JM, et al. Hypoxia-inducible factor 1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Pro Natl Acad Sci USA,1997;94:8104-8109
4喻晓蔚.缺氧诱导因子-1与肿瘤.中国肿瘤,2002,5(11)：286-288
5代志军,高洁,王西京,等.乳腺导管增生癌变过程中HIF-1α的表达变化及意义.中国肿瘤临床与康复,2007,14(1)：20-22.
6高勇强,谢文彪,谷依学,等.乳腺癌中HIF-1α, PCNA及Bcl-2的表达分析.南华大学学报,2006,34(2)：200-202.
7 Bos R,Zhong H, Hanmdan FC, et al. Levels of hypoxia inducible factor 1 alpha during breast carcinogenesis. Nat Cancer Inst Monogr,2001,93:309-314.
8. Zhang Q, Tang X, Lu Q, et al. Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia-and serum-induced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells. Mol Cancer Ther. 2006;5(5):1227-38.
9. Zhang CY, Zhang JX, Lu XT, et al, Effects of intermittent hypoxic exposure on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha and erythropoietin levels.Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2009;25(10):932-4.
10.Gort EH, Groot AJ, Derks van de Ven TL, et al. Hypoxia-inducible factor-1alpha expression requires PI 3-kinase activity and correlates with Aktl phosphorylati-on in invasive breast carcinomas. Oncogene.2006;25(45):6123-6127.
11.Thomas R, Kim MH,HIF-1 alpha:a key survival factor for serum-deprived prost-ate cancer cells. Prostate.2008;68(13):1405-15.
12.Petit T, Borel C, Ghnassia J P, et al. Chemotherapy response of breast can cer depe nds on HER-2 status an d an thracycline doseintensity in the neoadjuvant setting. Clin Cancer Res,2001,7(6):1577-1581.
13.SlamonDJ, Leyland JB, Shak S, et al. Use of chemotherapy plus a monoelonal antibody against HER2 for metastatic breast cancer that overxpresses HER2. N Engl J Med,2001,344(11):783-792.
14.Laughner E, Taghavi P, Chiles K, et al. HER2 signaling increases the rate of hypexia-inducible-la synthesis:novel mechan ism for HIF-1 mediated vascular endothelial growth factor Expression. Mole Cell Biol,2001,21(12):3995-4004
15.Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidyli-nositol-3 kinase/Akt in HER2 overexpressing cells. Cancer Res,2005,65:3257-3263.
16. Hao Ls, Zhu X, Qian K, et al. Expression of hypoxia-inducible factor 1 alpha in human breast carclnonla. West China Medical,2006,21(3):445-446.
17.王耕,乳腺癌组织中缺氧诱导因子-1α的表达.肿瘤防治研究.2006,6(33)：391-393
18.John R. Griffiths, Marion Stubbs Opportunities for studying cancer by metabolo-mics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Advan. Enzyme Regul.2003 (43):67-76
19 EH Gortl, AJ Grootl, TLP Derks van de Venl,et,alHypoxia-inducible factor-la expression requires PI 3-kinase activity and correlates with Aktl phosphorylation in invasive breast carcinomas. Oncogene,2006 (25),6123-6127
20. Yang ZF, Poon RT, To J, et al. The potential mh of hypoxia-inducible factor 1 alpha in tumor progression after hypoxia and chemotherapy in hepatocelular carcinoma. Cancer Res,2004,64(15):5496-5503
21.chemotherapy in hepatocelular carcinoma. Cancer Res,2004,64(15):5496-5503.
22. Mabjeesh NJ, Escuin D, La Vallee TM, et al.2ME2 inhibits tumor growth and angiogenesis by disrupting micmtubules and dysregulating HIF. Cancer Cell, 2003,3(4):363-375.
23.Yeo EJ, Chun YS, Cho YS, et al. YC-1:apotential anticancer drug Targeting hypoxia-inducible factor 1. J Nail Cancer Inst,2003,95(7):516-525.
24.Liu SC, Minton NP, Giaccia AJ, et al. Antican cereficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene Ther,2002,9(4):291-296.
24.Post DE, van Meir EG. A novel hypoxia-inducible factor(HIF) activated oncolytic adenovirus for cancer therapy. Oncogene,2003,22(14):2065-207
2. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents:defining priorities to reduce cancer dispariti-es in different geographic regions of the world. J Clin Oncol,2006,24(14):2137-2150.
3.张思维,陈万青,孔灵芝,等.中国部分市县1998-2002年恶性肿瘤的发病与死亡.中国肿瘤,2006,15(7)：430-448
4.陈可欣,何敏,董淑芬,等.天津市女性乳腺癌发病率死亡率和生存率分析.中华肿瘤杂志,2002,6(24)：573-575
5. Ranson M, Tian Z, Andronicos NM, et al. In vitro cytotoxicity of bismuth-213 (213Bi)-labeled-plasminogen activator inhibitor type 2 (alpha-PAI-2) on human breast cancer cells. Breast Cancer Res Treat.2002;71(2):149-59.
6.吴敏,张建新,庞利群.乳腺癌微转移的研究进展.江苏大学学报(医学版),2007；4(17)：366-367
7.王耕,乳腺癌组织中缺氧诱导因子-1α的表达.肿瘤防治研究.2006,6(33)：391-393
8. John R,Griffiths, Marion Stubbs Opportunities for studying cancer by metabolom-ics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Advan. Enzyme Regul.2003 (43):67-76
9. EH Gortl, AJ Groot 1, TLP Derks vande Venl,et al, Hypoxia-inducible f actor-1 a expression requires PI 3-kinase activity and correlates with Aktl phosphorylation in invasive breast carcinomas. Oncogene,2006 (25),6123-6127
10. Robey IF, Lien AD, Welsh SJ,et al, Hypoxia-Inducible Factor-la and the Glycolytic Phenotype in Tumors 1, Neoplasia.2005,4(7):324-330
11.马超,周庚寅,肖颖,等.HIF-1αP-gp在腋淋巴结阴性乳腺癌组织中的表 达及意义.中国肿瘤临床,2007：1(34)：10-13
12.孙媛媛,徐向英.缺氧诱导因子-1α和血管内皮生长因子在乳腺癌中的表达及相关性.实用肿瘤学杂志,2006：4(20)：282-282
1. T.Thorsen, S.J.Maerkl, S.R. Quake, Miero-fluidielarge-sealeintegration.Seienee, 2002,298:580-584.
2. Webb R H.Confoeal optieal mieroseopy. Rep. Prog. Phys.1996,59(3):427-471.
3. J.W.Grate, C.J.B.Lea, A.E.Jarrell, et al. Automated sample Preparation method for suspension arrays using renew able surfaeese Parations with multiplexed flow cytometry fluorescence detection. Analytical Chimica Acta.2003,478:85-98.
4. Hou J. Fluorescent microsPheres and their applications.The Latex Course.2000, 5:17-19.
5. R.Jerrold Fulton, Ralph L. McDad, Perry L.Smith, et al..Advanced multiplexed Analysis with theFloMetrix system.Clinieal Chemstry,1997,43:1749-1756.
6.Watanabe N, Miyamoto M, TokudaY, et al.Serum cerbB-2 in breast cancer patients. Acta Oncol,1994,33(8):901-904.
7. Slamon D J, Clark G M, Wong S G, et al.Human breast cancer:correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science, 1987,235(4785):177-182.
8. Quaranta M, Daniele A, Coviello M, et al. CerbB-2 protein level in tissue and sera of breast cancer patients:a possibly useful clinical correlation.Tumori,2006, 92(4):311-317.
9. Oakman C, Moretti E, Galardi F, et al, The role of topoisomerase Ⅱalpha and HER-2 in predicting sensitivity to anthracyclines in breast cancer patients.Cancer Treat Rev. Cancer Treat Rev.2009,35(8):662-667.
10. Fehm T,Gebauer G,JagerW. Clinical utility of serial serum c-erbB-2 determinations in the follow-up of breast cancer patients. Breast Cancer Res Treat,2002,75 (2):97-106.
11. Imoto S, Kitoh T, Hasebe T, Serum c-erbB-2 levels in monitoring of operable breast cancer patients.Jpn J Clin Oncol,1999,29 (7):336-339.
12. Fornier M, Risio M, Vanpoznak C, et al. HER2 testing and correlation with efficacy of trastuzumab therapy. Oncology (Huntingt),2002,16 (10):1340-1352.
13. Isola J J, Holli K, Oksa H, et al. Elevated erbB-2 oncoprotein levels in preoperative and follow-up serum samples define an aggressive disease course in patients with breast cancer. Cancer,1994,73(3):652-658.
14. Cook G B, Neaman I E, Goldblatt J L, et al.Clinical utility of serum HER-2/neu testing on theBayer Immuno 1 automated system in breast cancer.Anticancer Res, 001,21(2B):1465-1470.
15.Zhang Q,Tang Q,Lu Q, et al, Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia-and serum-induced HIF-1αlpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells. Mol Cancer Ther. 2006;5(5):1227-38.
16.Yamamoto Y,Ibsuki M, Okumura Y, et al. Hypoxia-inducible factor 1alpha is closely linked to an aggressive phenotype in breast cancer. Breast Cancer Res Treat.2008 Aug;110(3):465-475.
17. Bluff JE, Menakuru SR, Cross SS, et al, Angiogenesis is associated with the onset of hyperplasia in human ductal breast disease. Br J Cancer.2009 Aug 18;101(4):666-72.
18.Schindl M, Schoppmann SF, Samonigg H, et al;Austrian Breast and Colorectal Cancer Study Group.Clin Cancer Res. Overexpression of hypoxia-inducible factor-1alpha is associated with an unfavorable prognosis in lymph node-positive breast cancer.2002 Jun;8(6):1831-7.
19.Schindl M, Schoppmann SF, Samonigg H, et al. Overexp ression of hypoxia-inducible factor 1alpha is associated with an unfavorable prognose in lymph node-positive breast cancer. ClinCancer Res,2002,8 (6):1831-1837.
20.Bos R, vander Groep P, Greijer AE, et al. Levels of hypoxia-inducible factor-alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer,2003,97(6):1573-1581.
21. Chen HH,Su WC, Hypoxia-inducible factor-1 alpha correlates with MET and metastasis in node-negative breast cancer.Breast Cancer Res Treat.2007 Jun;103(2):167-75.
22. Bottaro DP, Liotta LA. Out of air is not out of action. Nature,2003,423 (6940): 593-595.
23. Bernards R. Cancer:cues for migration. N ature,2003,425(6955):247-248.
24. Kim KK, Lee JJ, Yang Y, et al. Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells.Carcinogenesis.2008;29(4):704-12. Epub 2008 Feb 6.
25.Bartlett JM, Munro A, Cameron DA, et al. Type 1 receptor tyrosine kinase profiles identify patients with enhanced benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial. J Clin Oncol.2008 N;26(31):5027-35.
26. Ma C, Zhou GY, Xiao Y, et al. Reversion the multidrug resistance of human breast carcinoma cells by RNA interference targeting HIF-1 alpha gene. Zhonghua Bing Li Xue Za Zhi.2006;35(6):357-60.
27.柳光宇,沈坤炜,邵志敏,等.乳腺癌组织缺氧与雌激素受体-α的关系.中国癌症杂志,2004,14(2)：127-130.
28.Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexp ressing cells. Cancer Res,2005,65 (8):3257-3263.
29. Giatromanolaki A, Koukourakis MI, Simopoulos C, et al, Cancer Biol Ther. Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original c-erbB-2/HIF1alpha phenotype,2007;6(2):153-5.
30. Benezra R, Henke E, Ciarrocchi A, et al, Induction of complete regressions of oncogene-induced breast tumors in mice. Cold Spring Harb Symp Quant Biol. 2005;70:375-81.
31. Li YM, Zhou BP, Deng J, et al, A hypoxia-independent6202hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells.Cancer Res.2005,15;65(8):3257-63.
32. Giatromanolaki A, Koukourakis MI, Simopoulos C, et al. c-erbB-2 related aggressiveness in breast cancer is hypoxia inducible factor-1alpha dependent.Clin Cancer Res.2004;10(23):7972-7.
33. Menendez JA, Vellon L, Oza BP, et al. Does endogenous fatty acid metabolism allow cancer cells to sense hypoxia and mediate hypoxic vasodilatation? Characterization of a novel molecular connection between fatty acid synthase (FAS) and hypoxia-inducible factor-1alpha (HIF-1alpha)-related expression of vascular endothelial growth factor (VEGF) in cancer cells overexpressing her-2/neu oncogene.J Cell Biochem.2005;94(5):857-63.
34. Howard EM, Lau SK, Lyles RH, et al, Expression of e-cadherin in high-risk breast cancer. J Cancer Res Clin Oncol.2005;131(1):14-8.
35. Laughner E, Taghavi P, Chiles K, et al, Mol Cell Biol. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.2001;21(12):3995-4004.
36. Matsumoto K, Arao T, Tanaka K, et al. mTOR signal and hypoxia-inducible factor-1 alpha regulate CD133 expression in8-Azaguaninecancer cells.Cancer Res.2009;69(18):7160-4.
37. Liu M, HowesA, Lesperance J, Antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer. Cancer Res,2005,65 (12):5325-5336
38. Shafee N, Kaluz S, Ru N, et al, PI3K/Akt activity has variable cell-specific effects on expression of HIF target genes, CA9 and VEGF, in human cancer cell lines.Cancer Lett.2009;282(1):109-15.
1. Taylor J, King R D, Fiehn O, et al. App lication ofmetabolomics to plant genotype discrimination using statistics and machine learning. Bioinform atics,2002,18 (10):241-248.
2. Reo N V. NMR2based metabolomics. D rug Chem Toxicol,2002,25 (4):375-382.
3.桑志红,杨松成.液相色谱-质谱联用技术及其在药物分析中的应用.解放军药学学报,1999,15(2)：28-31.
4:Miyoshi Y, Hamase K, Tojo Y, et al, Determination of D-serine and D-alanine in the tissues and physiological fluids of mice with various D-amino-acid oxidase activities using two-dimensional high-performance liquid chromatography with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877(24):2506-12.
5:Penteado JC, Rigobello-Masini M, Liria CW, et al, Fluorimetric determination of intra-and extracellular free amino acids in the microalgae Tetraselmis gracilis (Prasinophyceae) using monolithic column in reversed phase mode.J Sep Sci. 2009;32(15-16):2827-34.
6:Bhushan R, Kumar R. Analysis of multicomponent mixture and simultaneous enantioresolution of proteinogenic and non-proteinogenic amino acids by reversed-phase high-performance liquid chromatography using chiral variants of Sanger's reagent.Anal Bioanal Chem.2009;394(6):1697-705.
7:Giuntini F, Bourre L, Macrobert AJ, et al. Quantitative determination of 5-aminolaevulinic acid and its esters in cell lysates by HPLC-fluorescence.J Chromatogr B Analyt Technol Biomed Life Sci.2008;875(2):562-6.
8. Rahman MM, Takafuji M, Ihara H. Synthesis and assessment of molecular recognizability by RP-HPLC of an N-alkyl-beta-Ala-L-Phe-derived organic phase with self-assembling ability.Anal Bioanal Chem.2008;392(6):1197-208.
9. Bergero D, Assenza A, Schiavone A, et al. Amino acid concentrations in blood serum of horses performing long lasting low-intensity exercise. J Anim Physiol Anim Nutr (Berl).2005;89(3-6):146-50.
10. Krstanovic M, Brgles M, Halassy B, et al. Purification and characterization of 1,(l/d)-aminopeptidase from Guinea pig serum. Prep Biochem Biotechnol. 2006;36(2):175-95.
11. Hanko VP, Rohrer JS. Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection. Anal Biochem.2004;324(1):29-38.
12. Josemans AI, Zweygarth E. Amino acid content of cell cultures infected with Cowdria ruminantium propagated in a protein-free medium. Ann N Y Acad Sci. 2002;969:141-6.
13. Morikawa A, Hamase K, Inoue T, et al. Determination of free D-aspartic acid, D-serine and D-alanine in the brain of mutant mice lacking D-amino acid oxidase activity. J Chromatogr B Biomed Sci Appl.2001;757(1):119-25.
14.郑璐侠,邵泓,陈钢,等,13种氨基酸和牛磺酸的柱前衍生化HPLC测定.中国医药工业杂志,2008,39(8)：610-612。
15.Yang J,Xu G W,Kong H W,et al.Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer..J Chrom atogr,2002,780(1):27-33.
16.Zheng Y F,Xu G W,L iu D Y,et al..Study of urinary nucleosides as biological marker in cancer patients analyzed by micellar electrokinetic capillary chromatography.E ctrophoresis,2002,23(24):4104-4109.
17..Friedecky D,Adam T,Bartak P. Cap illary electrophoresis for detection of inherited disorders of purine and pyrim idine metabolism:A selective app roach.E lectrophoresis,2002,23(4):565-571.
18.Claudia C, BirarelliM, NolaM, et al. Automated high-perform-ance liquid chromatographic method for the determination of ho-mocysteine in p lasma samp les.J Chrom atogrA,1999,846 (122):93-100.
19. Ben-yoseeph O, Badar-Goffer R S, Morris PG, et al. Biochem J,1993,291 (Pt 3):915-919.
20.John R. Griffiths, Marion Stubbs Opportunities for studying cancer by metabolom-ics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Advan. Enzyme Regul.2003 (43):67-76
21.Griffiths J R, Stubbs M, Opportunities for studying cancer by metabolomics: preliminary observations on tumors deficient in hypoxia-inducible factor 1. Adv Enzyme Regul,2003,43:67-76.
22. Reo N V. NMR-based metabolomics. Drug Chem Toxicol,2002,25 (4):375-382.
23.Cascino A, Muscaritoli M, Cangiano C,et al.Plasma aminoacid imbalance in patients with lung and breast cancer. Anticancer Res.1995;15(2):507-510.
24. Davies NP, Wilson M, Natarajan K, et al. Non-invasive detection of glycine as a biomarker of malignancy in childhood brain tumours using in-vivo 1H MRS at 1.5 Tesla confirmed by ex-vivo high-resolution magic-angle spinning NMR. NMR Biomed.2010;23(1):80-7.
25. Choi BD, Archer GE, Mitchell DA, et al. EGFRvⅢ-targeted vaccination therapy of malignant glioma.Brain Pathol.2009 Oct;19(4):713-23.
26. LIUHong-Yan, LUGuo-Wei, WANGWei-Zhong. Changes in glycine content in mouse brain during hypoxic preconditioning.Acta Physiologica Sinica. 2001,53(6),461-464。
27. Lu J, Chen ZW. Isolation, characterization and anti-cancer activity of SK84, a novel glycine-rich antimicrobial peptide from Drosophila virilis. Peptides. 2010;31(1):44-50
28. Pfeiffer F, Betz H. Solubilisation of the glycine receptor from the rat spinal cord. Brain Res,1981,226:273-279.
29.Iimuro Y, Bradford BU, Yamashina S, et al. The glutathione precursor L-2-oxothiazolidine-4-carboxylic acid protects against liver injury due to chronic enteral ethanol exposure in the rat. Hepatology,2000,31:391-398.
30.Li X, Bradford BU, Wheeler MD, et al. Dietary glycine prevents peptidoglycan polysaccharide-induced reactive arthritis in the rat:role for glycine-gated chloride channel. Infect Immun,2001,69:5883-5891.
31.Wheeler MD, Rose ML, Yamashima S, et al. Dietary glycine blunts lung inflammatory cell influx following acute endotoxin. Am J Physiol Lung Cell Mol Physiol,2000,279:390-398.
32.Wheeler MD, Stachlewitz RF, Yamashina S, et al. Glycine!gated chloride channels in neutrophils attenuate calcium influx and superoxide production. FASEB J,2000,14:476-484.
33. Stachlewitz RF, Li X, Smith S, et al. Glycine inhibits growth of Tlymphocytes by an IL-2-independent mechanism. J Immunol,2000,164:176-182.
34. Serganov A, Patel DJ. Amino acid recognition and gene regulation by riboswit-ches. Biochim Biophys Acta. Biochim Biophys Acta.2009;1789(9-10):592-611
35.Wang CL, Teo KY, Han B. An amino acidic adjuvant to augment cryoinjury of MCF-7 breast cancer cells.Cryobiology.2008;57(1):52-9.
36.Richardson AD, Yang C, Osterman A, et al. Central carbon metabolism in the progression of mammary carcinoma.Breast Cancer Res Treat.2008;110(2):297-307.
37.Hensley K, Robinson KA, Gabbita SP, et al. Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med,2000,28:1456-1462.
38.Wang HD, Lu XX, Lu DX, et al. Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFalpha production in cardiomyocytes by activating a glycine receptor. Acta Pharmacol Sin.2009;30(8):1107-14.
39. Nakamura Y, Matsumoto S, Mochida T, et al. Glycine regulates proliferation and differentiation of salivary-gland-derived progenitor cells.Cell Tissue Res. 2009;336(2):203-12.
40.王亮,李保义,李庆瑞,胃癌组织氨基酸与肿瘤体积相关性研究。肠外与肠内营养,2000,(17)：41-44.
41.Yuan G, Nanduri J, Bhasker CR, et al. Ca2+/calmodulin kinase dependent activation of hypoxia inducible factor 1 transcriptional activity in cells subjected to intermittent hypoxia. J Biol Chem,2005,280(6):4321-4328.
42.Yuan G, Nanduri J, Khan S, et al. Induction of HIF-1alpha expression by intermittent hypoxia:involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR.J Cell Physiol.2008;217(3):674-85.
43. Lee SH, Heo JS, Han HJ. Effect of hypoxia on 2-deoxyglucose uptake and cell cycle regulatory protein expression of mouse embryonic stem cells:involvement of Ca2+/PKC, MAPKs and HIF-1alpha.Cell Physiol Biochem.2007;19(5-6):269-282.
44.Shen WG, Peng WX, Shao Y, et al. Localization and activity of calmodulin is involved in cell-cell adhesion of tumor cells and endothelial cells in response to hypoxic stress.Cell Biol Toxicol.2007;23(5):323-35.
45.Osada-Oka M, Takahashi M, Akiba S, et al, Eur J Pharmacol. Involvement of Ca2+-independent phospholipase A2 in the translocation of hypoxia-inducible factor-1 alpha to the nucleus under hypoxic conditions.2006;549(1-3):58-62.
46.Yuan G, Nanduri J, Bhasker CR, et al. Ca2+/calmodulin kinase-dependent activation of hypoxia inducible factor 30,6-Djhydroxycholanic Acidtranscription -al activeity in cells subjected to intermittent hypoxia. J Biol Chem.2005;280(6): 4321-4328.
47.Wollheim CB. Beta-cell mitochondria in the regulation of insulin secretion:a new culprit in type 2 diabetes. Diabetologia,2000,43:265-277.
48.Civekek VN, Deeney JT, Shalosky NJ,et al. Regulation of pancreatic β-cell mitochondrial metabolism:influence of Ca2+,substrate and ADP. Biochem J 1996, 318:615-621.
49.Comerford KM, Wallace TJ, Karhausen J, et al. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance(MDR1) gene.Cancer Res,2002,62(12):3387-94.
1. Jemal A, Siegel R, Ward E, et al. Cancer statistics,2007. CA Cancer J Clin,2007, 57(1):43-66.
2. Scheurle D,Jahanzeb M,Aronsohn RS,et al. HER-2/neu expression in archival non-small lung carcinomas using FDA-approved Hercerptin lest. Anticancer Res,2000,20(3B):2091.
3.周爱萍,乳腺癌靶向治疗:Herceptin的研究进展.国外医学肿瘤学分册,2000,8(4)：167.
4.Lufmer D,luke C,Possinger K, serum HER-2/neu in the management of breast cancer patients. Clinic Biochem,2003,36:233.
5.Griffiths J R, Stubbs M, Opportunities for studying cancer by metabolomics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Adv Enzyme Regul,2003,43: 67-76.
6.代志军,高洁,王西京,等.乳腺导管增生癌变过程中HIF-1α的表达变化及意义.中国肿瘤临床与康复,2007,14(1)：20-22.
7.Bos R, Zhong H, Hanmdan FC, et al. Levels of hypoxia inducible factor 1 alpha during breast carcinogenesis. Nat Cancer Inst Monogr,2001,93:309-314.
8.Chen HH, Su WC, Hypoxia-inducible factor-1αlpha correlates with MET and metastasis in node-negative breast cancer.Breast Cancer Res Treat.2007;103(2): 167-75.
9.王耕,乳腺癌组织中缺氧诱导因子-1α的表达.肿瘤防治研究2006,6(33)：391-393.
10. Hao Ls, Zhu X, Qian K, et al. Expression of hypoxia-inducible factor 1 alpha in human breast carclnonla. West China Medical,2006,21(3):445-446.
11. Bos R, Zhong H, Hanmdan FC, et al. Levels of hypoxia inducible factor 1 alpha during breast carcinogenesis. Nat Cancer Inst Monogr,2001,93:309-314.
12. Yang ZF, Poon RT, To J, et al. The potential mh of hypoxia-inducible factor 1 alpha in tumor progression after hypoxia and chemotherapy in hepatocelular carcinoma. Cancer Res,2004,64(15):5496-5503
13.Griffiths J R, Stubbs M, Opportunities for studying cancer by metabolomics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Adv Enzyme Regul,2003,43: 67-76.
14.Edelstein CL, Ling H, Gengaro PE, et al. Effeet of glycine on Prelethal and Postlethal increases in calpain activity in rat renal Proximal tubules. Kidney international,1997,52(5):1271-1278.
15.Wheeler M, Stachlewitz RF, Yamashina S, et al. Glycine-gated chloride channels in neutronphils attenuate calcium influx and superoxide Production. FASEB J,2000,14(3):476-484.
16.Yamashina S, Konno A, Wheeler MD, et al. Endothelial Cells contain a glycine-gated Chloride channel. Nutr Cancer,2001,40(2):197-204.
17.Griffiths J R, Stubbs M, Opportunities for studying cancer by metabolomics: preliminary observations on tumors deficient in hypoxia-inducible factor 1. Adv Enzyme Regul,2003,43:67-76.
18.Yuan G, Nanduri J, Bhasker CR, et al. Ca2+/calmodulin kinase dependent activation of hypoxia inducible factor 1 transcriptional activity in cells subjected to intermittent hypoxia. J Biol Chem,2005,280(6):4321-4328.
19.Edelstein CL, Ling H, Gengaro PE, et al. Effeet of glycine on Prelethal and Postlethal increases in calpain activity in rat renal Proximal tubules. Kidney international,1997,52(5):1271-1278.
20.Koukourakis M I, Simopoulos C, Polychronidis A, et al, The effect of trastuzumab/docatexel combination on breast cancer angiogenesis:dichotomus effect predictable by the HIFI alpha/VEGF pre-treatment status? Anticancer Res. 2003;23(2C):1673-80.
21.Bos R, vander Groep P, Greijer A E, et al. Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer.2003;97(6):1573-81.
22.Yen L, Benlimame N, Nie ZR, et al. Differential regulation of tumor angiogenesis by distinct ErbB homo and heterodimers. Mol Biol Cell. 2002;13(11):4029-44
23. Laughner E, Taghavi P, Chiles K, et al, HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1 alpha (HIF-1 alpha) synthesis:novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol. 2001;21(12):3995-4004.
24. Bos R, Zhong H, Hanrahan CF, et al. Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis.J Natl Cancer Inst.2001;93(4):309-14.
25. Giatromanolaki A, Koukourakis MI, Simopoulos C, et al, Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original c-erbB-2/HIF1alpha phenotype. Cancer Biol Ther.2007;6(2):153-5.
26.Petit T, Borel C, Ghnassia J P, et al. Chemotherapy response of breast cancer depe nds on HER-2 status an d an thracycline doseintensity in the neoadjuvant setting. Clin Cancer Res,2001,7(6):1577-1581
27.Laughner E, Taghavi P, Chiles K, et al. HER2 signaling increases the rate of hypexia-inducible-la synthesis:novel mechan ism for HIF-1 mediated vascular endothelial growth factor Expression. Mole Cell Biol,2001,21(12):3995-4004
28.Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidyli-nositol-3 kinase/Akt in HER2 overexpressing cells. Cancer Res,2005,65:3257-3263.
29. Gort EH, Groot AJ, Derks van de Ven TL, et al. Hypoxia-inducible factor-1alpha expression requires PI 3-kinase activity and correlates with Akt1 phosphoryla-tion in invasive breast carcinomas. Oncogene.2006;25(45):6123-6127.
30. Liu M, HowesA, Lesperance J, Antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer. Cancer Res,2005,65 (12):5325-5336
31. Kim KK, Lee JJ, Yang Y, et al. Macrophage inhibitory cytokine-1 activates AKT and ERX-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells. Carcinogenesis.2008;29(4):704-12.
32. Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells.Cancer Res.2005;65(8):3257-63.
33.Kim KK, Lee JJ, Yang Y, et al. Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells.Carcinogenesis.2008;29(4):704-12.
34.Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexp ressing cells. Cancer Res,2005,65 (8):3257-3263.
35. Li YM, Zhou BP, Deng J, et al, A hypoxia-independent6202hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells.Cancer Res.2005;65(8):3257-63.
36. Shafee N, Kaluz S, Ru N, et al, PI3K/Akt activity has variable cell-specific effects on expression of HIF target genes, CA9 and VEGF, in human cancer cell lines.Cancer Lett.2009;282(1):109-15.
37. Liao JY, Li LL, Wei Q, et al, Arch Biochem Biophys. Heregulinbeta activates store-operated Ca2+ channels through c-erbB2 receptor level-dependent pathway in human breast cancer cells.2007;458(2):244-52.
38. Feinmesser RL, Wicks SJ, Taverner CJ, et al, Biol Chem. Ca2+/calmodulin-dependent kinase Ⅱ phosphorylates the epidermal growth factor receptor on multiple sites in the cytoplasmic tail and serine 744 within the kinase domain to regulate signal generation.1999;274(23):16168-73.
39.Civekek VN, Deeney JT, Shalosky NJ, et al. Regulation of pancreatic β-cell mitochondrial metabolism:influence of Ca2+, substrate and ADP. Biochem J 1996,318:615-621.
40.Giatromanolaki A, Koukourakis MI, Simopoulos C, et al, Cancer Biol Ther. Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original c-erbB-2/HIF1alpha phenotype,2007;6(2):153-5.
41. Benezra R, Henke E, Ciarrocchi A, et al, Induction of complete regressions of oncogene-induced breast tumors in mice. Cold Spring Harb Symp Quant Biol. 2005;70:375-81.
42. Giatromanolaki A, Koukourakis MI, Simopoulos C, et al. c-erbB-2 related aggressiveness in breast cancer is hypoxia inducible factor-1alpha dependent. Clin Cancer Res.2004;10(23):7972-7.
43. Menendez JA, Vellon L, Oza BP, et al. Does endogenous fatty acid metabolism allow cancer cells to sense hypoxia and mediate hypoxic vasodilatation? Characterization of a novel molecular connection between fatty acid synthase (FAS) and hypoxia-inducible factor-1alpha (HIF-1alpha)-related expression of vascular endothelial growth factor (VEGF) in cancer cells overexpressing her-2/neu oncogene.J Cell Biochem.2005;94(5):857-63.
44. Howard EM, Lau SK, Lyles RH, et al. Expression of e-cadherin in high-risk breast cancer. J Cancer Res Clin Oncol.2005;131(1):14-8.
45. Laughner E, Taghavi P, Chiles K, et al, Mol Cell Biol. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.2001;21(12):3995-4004.
46. Yuan G, Nanduri J, Khan S, et al. Induction of HIF-1alpha expression by intermittent hypoxia:involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR.J Cell Physiol.2008;217(3):674-85.
47. Tajima N, Schonherr K, Niedling S,et al, Ca2+-activated K+ channels in human melanoma cells are up-regulated by hypoxia involving hypoxia-inducible factor-1alpha and the von Hippel-Lindau protein. J Physiol.2006 1;571(Pt 2):349-59.
1.Nicholls AW, Holmes E, Lindon J C, et al. Metabonomic investigations into hydrazine toxicity in the rat. Chem Res Toxicol.2001,14(8):975-987.
2.Art hur J M, Thongboonker DV, Scherzer JA, et al. Differential expression of proteins in renal cortex and medulla:a proteomic approach. Kidny Int,2002,62(8): 1314-1318
3.Nicholson J K, Lindon J C, Holmes E, et al. Metabonomics:understanding the metabolic responses of living systems to pathophysiological stimuli viamultivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica,1999,29(11):1181-1189
4.Waters NJ, Holmes E, Warterfield CJ, et al, NMR and pattern recognition studies on liver extracts and intact livers from rats treated with anaphthylisonthiocyanate. Biochem Pharmacol,2002,64:67
5.Tavlor J, King RD, Altmann T, et al. Application of metabolomics to plant genotype discrimination using statistics and machine learning. Bioinformatics, 2002,18(Suppl 2):S241-S248
6.朱勇飞,张天宝.代谢组学在毒理学研究中的应用.国外医学卫生学分册,2005,32(3)：156-159
7.Fiehn O. Metabolomics the link between genotypes and phenotypes. Plant Mol Biol,2002,48(1/2):155-171
8. Douglas B K.Metabolom ics and system s biology:making sense of the soup.Cur Opin in M-icrobiology,2004,7:296
9. Greenlee RT, Murray T, Bolden S, et al.Cancer statistics,2000. CA Cancer J Clin. 2000;50(1):7-33
10.Tiwari RC, Ghosh K, Jemal A, et al. A new method of predicting US and state -level cancer mortality counts for the current calendar year. CA Cancer J Clin. 2004;54(1):30-40.
11.Moysich KB, Menezes RJ, Baker JA, et al Environmental exposure to polychlori-nated biphenyls and breast cancer risk. Rev Environ Health.2002;17(4):263-77.
12. Bray GA. The underlying basis for obesity:relationship to cancer. J Nutr. 2002;132(11 Suppl):3451S-3455S.
13.McTiernan A, Rajan KB, Tworoger SS, et al.Adiposity and sex hormones in postmenopausal breast cancer survivors. J Clin Oncol.2003;21(10):1961-6.
14.Key T, Appleby P, Barnes I, et al. Endogenous sex hormones and breast cancer in postmenopausal women:reanalysis of nine prospective studies. J Natl Cancer Inst.2002;94(8):606-16.
15.Klymachyov A, Dalai N.Spinning crystals leads to significant enhancement in 13C spectral resolution in MAS experiments on organic compounds:a new aid in studying phase transitions. Solid State Nucl Magn Reson.1997;9(1):85-9.
16. Castrillo JI, Hayes A, Mohammed S, et al. An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry. Phytochemistry.2003;62(6):929-37.
17. Potts BC, Deese AJ, Stevens GJ,et al.NMR of biofluids and pattern recognition: assessing the impact of NMR parameters on the principal component analysis of urine from rat and mouse. J Pharm Biomed Anal.2001;26(3):463-76.
18. Beckwith-Hall BM, Brindle JT et al.Application of orthogonal signal correction to minimise the effects of physical and biological variation in high resolution 1H NMR spectra of biofluids. Analyst.2002;127(10):1283-8.
19. Daykin CA, Corcoran O, Hansen SH et al.Application of directly coupled HPLC NMR to separation and characterization of lipoproteins from human serum. Anal Chem.2001;73(6):1084-90.
20.Griffin JL, Williams HJ, Sang E,et al.Metabolic profiling of genetic disorders:a multitissue (1)H nuclear magnetic resonance spectroscopic and pattern recogni-tion study into dystrophic tissue. Anal Biochem.2001;293(1):16-21.
21.Brindle JT, Antti H, Holmes E, et al.Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nat Med.2003;9(4):477.
22. Waters NJ, Holmes E, Waterfield CJ, et al.NMR and pattern recognition studies on liver extracts and intact livers from rats treated with alpha-naphthylisothiocya-nate. Biochem Pharmacol.2002;64(1):67-77
23.Griffin J L,Shockcor J P. Metabolic profiles of cancer cells. Nat Rev Cancer.2004 Jul;4(7):551-61
24.Cheng LL, Chang IW, Smith BL, et al.Evaluating human breast ductal carcinomas with high-resolution magic-angle spinning proton magnetic resonance spectroscopy. J Magn Reson.1998;135(1):194-202.
25. Fiehn O. Metabolomics-the link between genotypes and phenotypes. Plant Mol Biol.2002;48(1-2):155-71.
26. Mackinnon WB, Barry PA, Malycha PL, et al.Fine-needle biopsy specimens of benign breast lesions distinguished from invasive cancer ex vivo with proton MR spectroscopy. Radiology.1997;204(3):661-6.
27.许国旺,杨军.色谱,2003,21(4)：316-320.
28.Leach MO, Verrill M, Glaholm J, et al.Measurements of human breast cancer us-ing magnetic resonance spectroscopy:a review of clinical measurements and a report of localized 31P measurements of response to treatment. NMR Biomed. 1998;11(7):314-40.
29. Merchant TE, Kasimos JN, Vroom T, et al.Malignant breast tumor phospholipid profiles using (31)P magnetic resonance. Cancer Lett.2002;176(2):159-67.
30. Barzilai A, Horowitz A, Geier A, et al.Phosphate metabolites and steroid horm-one receptors of benign and malignant breast tumors. A Nuclear Magnetic Reso-nance study. Cancer.1991;67(11):2919-25.
31. Singer S, Souza K, Thilly WG. Pyruvate utilization, phosphocholine and adeno-sine triphosphate (ATP) are markers of human breast tumor progression:a 31P-and 13C-nuclear magnetic resonance (NMR) spectroscopy study. Cancer Res. 1995;55(22):5140-5.
32. Rivenzon-Segal D, Margalit R, Degani H.Glycolysis as a metabolic marker in ort-hotopic breast cancer, monitored by in vivo (13)C MRS. Am J Physiol Endocrinol Metab.2002;283(4):E623-30.
33. Katz-Brull R, Seger D, Rivenson-Segal D, et al.Metabolic markers of breast cancer:enhanced choline metabolism and reduced choline-ether-phospholipid synthesis. Cancer Res.2002;62(7):1966-70.
34. Yu JX, Kodibagkar VD, Liu L, et al.A 19F-NMR approach using reporter mole-cule pairs to assess beta-galactosidase in human xenograft tumors in vivo. NMR Biomed.2008;21(7):704-12.
35. Holmberg L, Jakobsson U, Berglund A, et al.Failure to detect early breast cancer using in vitro nuclear magnetic resonance spectroscopy of plasma. Br J Cancer. 1993;68(2):389-92.
36. Raffelt K, Moka D, Sullentrop F,et al.Systemic alterations in phospholipid con-centrations of blood plasma in patients with thyroid carcinoma:an in-vitro (31)-P high-resolution NMR study. NMR Biomed.2000;13(1):8-13.
37. Boss EA, Moolenaar SH, Massuger LF, et al.High-resolution proton nuclear magnetic resonance spectroscopy of ovarian cyst fluid. NMR Biomed.2000; 13 (5):297-305.
38. H.S.Shaw,M.J.Ellis, Letrozole in the treatment of breast cancer. Expert Opin Pharmacother.2002;3(5):607-17.
39. Glunde K, Ackerstaff E, Natarajan K, et al.Real-time changes in 1H and 31P NMR spectra of malignant human mammary epithelial cells during treatment with the anti-inflammatory agent indomethacin. Magn Reson Med.2002;48(5) :819-25.
40. Lonning PE, Geisler J, Johannessen DC, et al.Pharmacokinetics and metabolism of formestane in breast cancer patients. J Steroid Biochem Mol Biol.2001;77(1): 39-47.
41. Odunsi K, Wollman RM, Ambrosone CB, et al.Detection of epithelial ovarian cancer using 1H-NMR-based metabonomics. Int J Cancer.2005;113(5):782-8.
42. Whitehead TL, Monzavi-Karbassi B, Jousheghany F,et al.1H-NMR metabolic markers of malignancy correlate with spontaneous metastases in a murine mam-mary tumor model. Int J Oncol.2005;27(1):257-63.
43. Stanley EG, Bailey NJ, Bollard ME,et al.Sexual dimorphism in urinary metabo-lite profiles of Han Wistar rats revealed by nuclear-magnetic-resonance-based metabonomics. Anal Biochem.2005;343(2):195-202.
44. Slim RM, Robertson DG, Albassam M,et al. Effect of dexamethasone on the met-abonomics profile associated with phosphodiesterase inhibitor-induced vascular lesions in rats. Toxicol Appl Pharmacol.2002;183(2):108-9.
45. Lenz EM, Bright J, Wilson ID, Hughes A,et al. Metabonomics, dietary influenc-es and cultural differences:a 1H NMR-based study of urine samples obtained from healthy British and Swedish subjects. J Pharm Biomed Anal.2004;36(4): 841-9.
46. Tolstikov VV, Lommen A, Nakanishi K,et al. Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics. Anal Chem.2003 Dec 1;75(23):6737-40.
47. Sugimoto M, Kikuchi S, Arita M,et al. Large-scale prediction of cationic metabo -lite identity and migration time in capillary electrophoresis mass spectrometry using artificial neural networks. Anal Chem.2005;77(1):78-84.
48. Pham-Tuan H, Kaskavelis L, Daykin CA, et al. Method development in high-per-formance liquid chromatography for high-throughput profiling and metabonom-ic studies of biofluid samples. J Chromatogr B Analyt Technol Biomed Life Sci. 2003;789(2):283-301.
49.桑志红,杨松成.液相色谱-质谱联用技术及其在药物分析中的应用.解放军药学学报,1999,15(2)：28-31.
50.Yang J,Xu G W,Kong H W,et a.lA rtificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer..J Chrom atogr,2002,780(1):27-33.
51.Zheng Y F,Xu G W,L iu D Y,et al. Study of urinary nucleosides as biological marker in cancer patients analyzed by micellar electrokinetic capillary chromatography.E ctrophoresis,2002,23(24):4104-4109.
52.Friedecky D,Adam T,Bartak P.Cap illary electrophoresis for detection of inherited disorders of purine and pyrim idine metabolism:A selective app roach.E lectrophoresis,2002,23(4):565-571.
53. Sitter B, Sonnewald U, Spraul M, et al. High-resolution magic angle spinning MRS of breast cancer tissue. NMR Biomed.2002 Aug;15(5):327-37.
54. Plumb RS, Stumpf CL, Gorenstein MV, et al. Metabonomics:the use of electro-spray mass spectrometry coupled to reversed-phase liquid chromatography sho-ws potential for the screening of rat urine in drug development. Rapid Commun Mass Spectrom.2002;16(20):1991-6.
55.Xiang R, Shi Y, Dillon DA, et al.2D LC/MS analysis of membrane proteins from breast cancer cell lines MCF7 and BT474. J Proteome Res.2004;3(6):1278-1283.
56.Zang L,Palmer Toy D,Hancock WS, et al.Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 160/180 isotopic labeling. J Proteome Res.2004;3(3):604-12.
57.Chong BE, Yan F, Lubman DM,et al. Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates:a novel two-dimensional liquid chromatography/mass spectrometry method.Rapid commun Mass spectrum.2001;15(4):291-6.
58.Mbeunkui F,Metge BJ,Shevde LA,et al.Identification of differentially secreted biomarkers using LC-MS/MS in isogenic cell lines representing a progression of breast cancer. J Proteome Res.2007;6(8):2993-3002.
59.Byun JA,Lee SH, Jung BH,et al,Analysis of polyamines as carbamoyl derivatives in urine and serum by liquid chromatography-tandem mass spectrometry.Biomed chromatogr.2008;22(1):73-80
60.Murph M, Tanaka T,Pang J, et al. Liquid chromatography mass spectrometry for quantifying plasma lysophospholipids:potential biomarkers for cancer diagnosis. Methods enzymol.2007;433:1-25.
61.Whiteaker JR,Zhang H,Zhao L,et al.Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer. J Proteome Res.2007;6(10):3962-75.
62. Qin F, Zhao YY, Sawyer MB,et al. Hydrophilic interaction liquid chromatography-tandem mass spectrometry determination of estrogen conjugates in human urine. Anal Chem.2008;80(9):3404-11.
63.沈朋,曾真,程翼宇.乳腺癌血清蛋白质组与尿代谢物组协同分析研究.中华检验医学杂志.2005,28(7)：156-159
64.Shi Y,Xiang R,Horvath C, et al. Quantitative analysis of membrane proteins from breast cancer cell lines BT474 and MCF7 using multistep solid phase mass tagging and 2D LC/MS.J Proteome Res.2005 4(4):1427-33.
1 Semenza GL, ADmi F, Booth G, et al. Structural andfunctionalan alysis of hypoxia-nducible factor 1. Kidney Int,1997;51:553-55
2 Minet E, Michel G, Remacle P, et al. Role of HIF-1 as a transciption factor involved in embryonic development, ca/leer progression and apoptosis. Int J Mol Med,2000;5:253-259
3 Maxwell PH, Dachs GU, Gleadle JM, et al. Hypoxia-inducible factor 1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Pro Natl Acad Sci USA,1997;94:8104-8109
4喻晓蔚.缺氧诱导因子-1与肿瘤.中国肿瘤,2002,5(11)：286-288
5代志军,高洁,王西京,等.乳腺导管增生癌变过程中HIF-1α的表达变化及意义.中国肿瘤临床与康复,2007,14(1)：20-22.
6高勇强,谢文彪,谷依学,等.乳腺癌中HIF-1α, PCNA及Bcl-2的表达分析.南华大学学报,2006,34(2)：200-202.
7 Bos R,Zhong H, Hanmdan FC, et al. Levels of hypoxia inducible factor 1 alpha during breast carcinogenesis. Nat Cancer Inst Monogr,2001,93:309-314.
8. Zhang Q, Tang X, Lu Q, et al. Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia-and serum-induced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells. Mol Cancer Ther. 2006;5(5):1227-38.
9. Zhang CY, Zhang JX, Lu XT, et al, Effects of intermittent hypoxic exposure on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha and erythropoietin levels.Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2009;25(10):932-4.
10.Gort EH, Groot AJ, Derks van de Ven TL, et al. Hypoxia-inducible factor-1alpha expression requires PI 3-kinase activity and correlates with Aktl phosphorylati-on in invasive breast carcinomas. Oncogene.2006;25(45):6123-6127.
11.Thomas R, Kim MH,HIF-1 alpha:a key survival factor for serum-deprived prost-ate cancer cells. Prostate.2008;68(13):1405-15.
12.Petit T, Borel C, Ghnassia J P, et al. Chemotherapy response of breast can cer depe nds on HER-2 status an d an thracycline doseintensity in the neoadjuvant setting. Clin Cancer Res,2001,7(6):1577-1581.
13.SlamonDJ, Leyland JB, Shak S, et al. Use of chemotherapy plus a monoelonal antibody against HER2 for metastatic breast cancer that overxpresses HER2. N Engl J Med,2001,344(11):783-792.
14.Laughner E, Taghavi P, Chiles K, et al. HER2 signaling increases the rate of hypexia-inducible-la synthesis:novel mechan ism for HIF-1 mediated vascular endothelial growth factor Expression. Mole Cell Biol,2001,21(12):3995-4004
15.Li YM, Zhou BP, Deng J, et al. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidyli-nositol-3 kinase/Akt in HER2 overexpressing cells. Cancer Res,2005,65:3257-3263.
16. Hao Ls, Zhu X, Qian K, et al. Expression of hypoxia-inducible factor 1 alpha in human breast carclnonla. West China Medical,2006,21(3):445-446.
17.王耕,乳腺癌组织中缺氧诱导因子-1α的表达.肿瘤防治研究.2006,6(33)：391-393
18.John R. Griffiths, Marion Stubbs Opportunities for studying cancer by metabolo-mics:preliminary observations on tumors deficient in hypoxia-inducible factor 1. Advan. Enzyme Regul.2003 (43):67-76
19 EH Gortl, AJ Grootl, TLP Derks van de Venl,et,alHypoxia-inducible factor-la expression requires PI 3-kinase activity and correlates with Aktl phosphorylation in invasive breast carcinomas. Oncogene,2006 (25),6123-6127
20. Yang ZF, Poon RT, To J, et al. The potential mh of hypoxia-inducible factor 1 alpha in tumor progression after hypoxia and chemotherapy in hepatocelular carcinoma. Cancer Res,2004,64(15):5496-5503
21.chemotherapy in hepatocelular carcinoma. Cancer Res,2004,64(15):5496-5503.
22. Mabjeesh NJ, Escuin D, La Vallee TM, et al.2ME2 inhibits tumor growth and angiogenesis by disrupting micmtubules and dysregulating HIF. Cancer Cell, 2003,3(4):363-375.
23.Yeo EJ, Chun YS, Cho YS, et al. YC-1:apotential anticancer drug Targeting hypoxia-inducible factor 1. J Nail Cancer Inst,2003,95(7):516-525.
24.Liu SC, Minton NP, Giaccia AJ, et al. Antican cereficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene Ther,2002,9(4):291-296.
24.Post DE, van Meir EG. A novel hypoxia-inducible factor(HIF) activated oncolytic adenovirus for cancer therapy. Oncogene,2003,22(14):2065-207