摘要
前言
宫颈癌是女性生殖系统常见的肿瘤,其发病率居恶性肿瘤之首,是女性生殖系统恶性肿瘤死亡的主要原因之一,所以宫颈癌的早期诊断有着重要的意义。本实验应用瘤细胞悬液移植法,在具有正常免疫功能的大鼠体内建立大鼠宫颈癌移植瘤动物模型,为宫颈肿瘤的早期诊断提供前期的动物实验依据。探讨大鼠宫颈癌移植肿瘤组织中的有丝分裂原激活蛋白激酶P38的表达及其临床意义;研究P38的高表达与淋巴结转移的关系。同时应用新一代超声造影剂声诺维观察造影对实验兔肌肉内人宫颈癌移植肿瘤的血流灌注显像的影响,评价声诺维(SonoVue)在超声显像中的造影增强作用,探讨及检验不同造影剂剂量在二次谐波显像中对兔宫颈癌移植瘤造影效果的影响与变化规律,为临床肿瘤诊断和鉴别诊断提供依据。
方法
第一部分:
体外培养HELA细胞,采用细胞记数法观察细胞生长情况。将对数生长期的HELA按2.5×10~7、2.5×10~6、2.5×10~5分别接种于大鼠的背部皮下、腹腔内、静脉注射,建立宫颈癌移植瘤动物模型,并于接种后观察成瘤率、肿瘤生长及播散情况及动物生存期,于种植后第7D、10D、14D、17D、21D行超声检查,然后等到实验鼠自然死亡,并对肿瘤进行病理学检查,确定肿瘤的存在及性质。
第二部分:
应用免疫组织化学方法和Western-blot方法检测人宫颈癌鼠移植瘤及其癌旁正常组织中P38及磷酸化P38的蛋白表达情况。
(一)免疫组化法
1.按照试剂盒说明书步骤抗p38免疫组化染色。切片常规脱蜡致水,高温高压抗原修复,一抗4℃孵育过夜,DAB显色,苏木素复染。PBS代替一抗作为阴性对照,用已知宫颈癌阳性片做阳性对照。
2.结果判定:在双盲法下进行,以肿瘤细胞内出现黄色或棕黄色颗粒为阳性表达。每张切片在光、镜下随机选取5个视野,每个视野计数100个瘤细胞,着色强度分为无色(一)为0分,浅黄色(+)为1分,黄色及棕黄色(++)为2分;阳性细胞数＜10%为0分,10%—50%为1分,＞50%为2分。两项得分相乘结果≥2记为阳性。
(二)Western blot法
取-70°保存的移植瘤及癌旁正常组织约100mg加入预冷的蛋白裂解液,剪碎,匀浆超声处理,高速低温离心提取上清为细胞总蛋白。电泳(12%SDS-PAGE凝胶)、转印,5%脱脂奶粉封闭液封闭1h,一抗4℃孵育过夜,与相应二抗室温下孵育2h,曝光,双蒸水冲洗,干燥,避光保存,结果经自动电泳凝胶成像分析仪(chemilmager 5500,Alpha Innotech,USA)采集,进行灰度值测定。
第三部分:
建立兔宫颈癌移植瘤模型十只。彩超每周观察肿瘤生长情况,共10周,于1～4周测量血流动力学参数;同时,患有宫颈癌移植瘤的兔分别经耳缘静脉团注法注射声学造影剂0.2、0.4、0.6ml/kg,然后用二次谐波显像观察肿瘤造影效果;应用彩色多普勒、能量多普勒观察兔肌肉层内移植瘤血流灌注情况并定量分析。
结果
第一部分:
在体外培养条件下的HELA细胞生长迅速,将不同数量的HELA细胞接种大鼠不同部位后,成瘤率为67%,迅速发生腹腔播散并产生大量腹水,其生物学行为与人宫颈肿瘤相似。肿瘤细胞负荷增加,动物生存期明显缩短(p＜0.01)。三组模型的组织病理学检查无明显差别。肿瘤的内部回声可分为高回声、等回声、低回声三类,随着肿瘤的生长其回声类型有所转变;鼠的人宫颈癌移植瘤的生长状况、回声类型及血供变化的动态演化大体符合宫颈癌的一般超声表现特点。该种植瘤的增长是进行性的,超声征象随肿瘤的进展而逐步体现,基本符合肿瘤侵袭的定义特征,该动物模型可用于超声对宫颈癌恶性生物学行为的观察。
第二部分:
P38在宫颈癌移植瘤组织中的表达阳性率为95.5%,在癌旁正常组织中的表达为33.1%。P38在宫颈癌移植瘤中的表达明显高于癌旁组织,有显著的统计学差异(P＜0.01)。P38的表达定位于胞质中,胞核中无表达;P38的表达与病程、肿瘤大小、肿瘤位置无明显相关。P38的表达与组织分化程度及有无淋巴结的转移有显著差异(P＜0.01)。
第三部分:
在兔的宫颈癌移植瘤模型中:PDI可探及星点状、短棒状和条状血流,各时期RI和PI值无显著性差异。造影后增强了肌肉内移植瘤的血管显示率及血流信号强度,肿瘤造影表现为快速增强,缓慢减退,持续时间较长的特点;新型声学造影方法可以明显增强肿瘤的二维超声影像。
结论
在具有正常免疫功能的大鼠体内成功建立的宫颈癌移植瘤模型能较好地模拟人自发宫颈癌的生物学特征,有助于了解宫颈癌生物学及免疫学特性,可作为良好的肿瘤模型用于宫颈癌的免疫治疗研究及进行影像学的研究。该种植瘤超声征象随肿瘤的进展而逐步体现,基本符合肿瘤侵袭的定义特征,该动物模型可用于超声对宫颈恶性肿瘤生物学行为的观察。本实验虽然属于临床前期的动物研究,但肿瘤直径基本≤3cm,其超声常规指标的动态观测将有助于临床早期宫颈肿瘤的诊断与鉴别。声学造影提高了兔肌肉内宫颈癌移植瘤血流灌注的显示,增强其血流信号,造影显像优于彩色多普勒显像。P38在宫颈肿瘤的细胞中呈高表达,并与病程进展密切相关,与宫颈癌的发生、发展及转移密切相关。
Objective
Cervical carcinoma is a common malignant tumor.To establish an animal model of cervical cancer in immunocompetent rat and to study its biological character.Based on pervious study,the purpose of this study is to found Hela mice tumor xenograft model and study the,through which to provide a way to prevent and cure the cervical cancer in clinic.To explore the regularity of the ultrasonographic dynamic changes such as the size,echo-intensity and edge echo-pattern of cervical tumor during its growth. To offer the animal experimental proves of the preclinical phase for the early diagnosis and treatment of cervical carcinoma.To investigate P38MAPK protein expression and its clinical significance in the cervical transplanted carcinoma of rat.In the meanwhile, to assess the blood perfusion of tumors in rabbit muscles with perfluorocarbon contrast flow imaging and high-frequency ultrasound.
Methods
Part one:
Hela was frozen in liquid nitrogen were revived in routine method and inoculated in RPMI-1640 Blended with 10%foetus cattle serum.HELA cells 2×10~7,2×10~6,2×10~5 was injected intraperitoneally,under skin,and vein.Human cervical cancer Hela cells were implanted into the mice to get the tumor model of HELA cell line.The transplanted tumors were observed every two days.The ultrasonographic observation was performed on the 7,10,14,21 and 30 day after implantation.The survivals were put to death and necropsy.The section preparations were stained with HE method.
Part two:
Hela was frozen in liquid nitrogen were revived in routine method and inoculated in RPMI-1640 Blended with 10%foetus cattle serum.2×10~5 was injected under skin. Immunohistochemical assay and Westem Blot was performed to study the expression and location of P38.Subcutaneously injected with PBS as a control group in 5 mice.
Part Three:
Ten rabbits model with cervical transplanted tumors in muscle were examined, and they were assessed by high-frequency color Doppler ultrasound every week for ten weeks.SonoUve was administered respectively to ten rabbits through ear veins at 0.2, 0.4,0.6 ml/kg,and then imaged with second harmonic imaging.After intravenous administration of contrast agent,Color Doppler imaging or Power Doppler imaging were used to evaluate the blood perfusion of tumors in muscle.
Results
Hela cells grew progressively after injected into rats intraperitioneally,formed gross peritoneal dissemination and malignant hemorrhagicascit-es in a manner typical for human cervical carcinomas.Animal survival time decreased significantly with large Hela burden(p＜0.01).Pathological analysis and tissue culture confirmed the existence of the carcinoma tissue.The implant,The eho-intensity of the tumor can be divided into three patterns such as hyperecho,isoecho and hypoecho.The observed rate of abnormal vessels in the tumor increased as the implantation time elongated and the tumor enlarged.The observed time of arterial blood was earlier than that of venous blood.The effective factors of histopathology mostly were necrosis,fiber and sinusoid. In two -dimensional images,tumors were small and poor echo one week after implantation.Necrosis and liquefaction appeared could be found later at the 6-7 week cavum formed.Color Doppler flowing showed dot,branch,or line flow signals and the degree of blood abundance was from 0-Ⅲgrades,while PI and RI between phrases had no significant difference,there were significant improvements in detecting neovascularity of tumor in muscle at all contrast agent dosage levels and the intensity lasted a long period.P38 was mainly expressed in the cell cytoplasm and none nuclei of cervical cells and neutrophil and lymphcytes.Positive rate of p38MAPK protein expression was 70%in carcinoma tissues,33.1%in its paraneoplastic tissue,and there is a significant correlation between the expression of P38 with the stages of cervical cancer,histology grade,and local lymph node ivolvement,but not with time,site or size of tumor.
Conclusions
A positive cervical cancer model has been successfully established in rats which have immunocompetent function that simulates preferably the biological behavior of human spontaneous cervical carcinoma.And is an optimal animal model for cervical cancer immunological therapy research.The ultrasonographic signs such as the size, echo -intensity,edge echo-pattern and blood flow changes of tumor during its growth were coincident to clinical cervical tumor.The gradual growth and gradual behavior of ultrasonography were essentially coincident to the defintive characteristics of tumor invasion.This animal model can be used to observe the malignant biological behaviors. The sizes of most tumors were less than 3cm,therefore the dynamic observation of the ultrasonographic roution parameters is useful to the diagnosis and odentification of the clinical little tumor.P38 is overexpressed in cervical cancer cells.The expression of P38 has a significant correlation with the stages of cervical cancer,p38 may play an important role in the occurrence,development and metastasis of human cervical carcinoma.The contrast agent is able to detect clearly the tumor vessels which cannot be shown with conventional color flow imaging.Moreover,the perfluorocarbon contrast agent can maintain the visuality of the vessels for a longer period.
引文
1 李雁,汤猷.我国肝癌模型研究的历史和现状.中华实验外科杂志,2001,18(5):479-480
2 凌瑞,沉江浩,姚青,等.兔转移性乳腺癌模型:肿瘤生长及转移.中国癌症杂志.2005,151:9-11
3 Kaufnann Y,Luo S,Johnson A,et al.Tining of oralglutamine on DMBA-induced tumori genesis.J Surg Res,2003,111(1):158-165
4 Beckhove P,Schutz F,Diel II,et al.Efficient engrafinent of human primary brestcancer transplants in nonconditioned NOD/Scid mice.Int J Cancer,2003,105(4):444-453
5 Christofferson RH,Skoldenberg EG,Nillson BO.Tumor growh in a defend microcirculation MPMIS,1998,105(6):467-49
6 Stratton JA,Micha JP,Rettenmaier MA,et al.Chemosensitivity testing of nonsolid tumors by the subrenal-capsule implant assay.Gynecol Oncol,1984,17(2):185-188
7 Shao GL,Zhou KR,Wang JH,et al.Improvement in making rabbit model bearing VX2liver tumor for interventional experimental study and CT evaluation of tumor.Clin Radiol,2000,19(10):653-654
8 Rodriguer Burford CMPH,Bames MN,Berry W,et al.Immunor histochemical expression of molecular markers in an ovian model:a potential model for preclinical evaluation of agents for ovarian cancer chemoprevention.GynecolOncol,2001,81:373-379
9 王文见,欧阳茂.高强度聚焦超声对大鼠肝癌及外周血淋巴细胞功能的影响.中华理疗杂志,2001,24(1):26-28
10 祝金泉,张韵和.用免疫抑制大鼠建立人胃癌模型的初步探讨.中华消化杂志,1999,10(5):280-281
11 Rose GS,Tocco LM,Cranger GA,et al.Development and characterization of a clini cally useful animal model of epithelial ovarian cancer in the Fischer 344 rat.Am J Obstet Gynecol,1996,175:593-599
12 崔恒,李艺.免疫重建荷人卵巢癌-严重联合免疫缺陷小鼠模型的建立.北京医科大学学报,2000,32:488-492
13 梁华茂,姜洁.Fischer 344大鼠上皮性卵巢癌动物模型的建立及其生物学特性.现代妇产科进展,2003,12(5):367-369
14 程文,张青萍. 超声监护下免疫抑制大鼠肝癌动物模型的建立.中国医学影像技术,2003, 19 (7):840-842
15 Fidler II.Critical factors in the biology of human cancer metastas is:twenty-eight GHA clowesmemorial lecture. Cancer Res, 1990, 50:6130-6135
16 Fwrukawa T, Fu X, K ubuta T. Nude mouse metastatic models of human stomach cancer constructed using orthotopic implantation of histologically intact tissue. Cancer Res, 1993, 53:1204-1208
17 Fu X, Hoffman RM. Human ovarian carcinoma metastatic models constructed in mude mice by orthotopic transplantation of histologically intact patient specimens. Anticancer Res, 1993,13:283-285
18 Welch DR. Technical considerations for studying cancer metastasis in vivo. Clin Exp Metastasis, 1997,15:272-306
19 Xu SH, Qiang LJ, Mou HZ, et al. Establishment of a highly metastatic human ovarian cancer cell line.Exp Clin Cancer Res, 1999,18:233-236
1 Frasch SC,Nick JA,Fadok VA,et al.P38 mitogenactivated protein kinase-dependent and independent in tracellular signal transduction pathways leading to apoptosis in human neutrophils.J Biol Chem,1998,273(14):8389-8391
2 Walshi JM.Cervical cancer:developments in screening and evaluation of the abn -ormal Pap smear.West J Med,1998,169:304-310
3 Zur Hausen H.Papillomaviruses in human cancers.Proc Assoc Am Physicians,1999,111:581-587
4 Stoler MH.Human papillomaviruses and cervical neoplasia:A model for carcinogen -esis.Int J Gynecol Pathol,2000,19:16-24
5 Porreco R,Penn I,Droegemueller W,et al.Gynecologic malignancies in immunosuppr -essed organ homograft recipients.Obstet Gynecol,1985,459(4):359-364
6 张剑,王树俊,裘宋良,等.EEK1和P38在食管鳞癌组织中的表达.肿瘤基础与临床,2006,19(3):177-179
7 Xia Z,Dickens M,Raingeaud J,et al.Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.Science,1995,270(5240):1326-1328
8 Karin M.The regulation of AP21 activity by mitogen-activated protein kinase.J Biol Chem,1995,270(16):16483-16486
9 Han J,Lee JD,Bibbs L,et al.A Map kinase treated by endotoxin and hyperos molarity in mammalian cells.Science,1994,265(5173):808-811
10 Huang S,New L,Pan ZX,et al.Urokinase plasmvnitogon altinator lurokinase - speci -fis surface receptor expression and matnix invasion by breast cancer cells requires constitutive p38 mitogen- activated protein kinase activity.J Biol Chem,2000,275(16):12266-12272
11 Kim MS,Lee EJ,Kim HR,et al.p38 kinase is a key signaling molecule for H-ras -induced cell motility and invasive phenotype in human breast epithelial cells.Cancer Res,2003,63(17):5454-5461
12 李艳波,李为民,韩君勇.P38信号通路在人脐静脉内皮细胞表达环氧化酶2中的作用.医学研究生学报,2005,18(6):490-492
13 Vadlamudi R,Adam L,Talukder A,et al.Scrine phiosphorylaton of paxiuin by heregnlin-β:role of p38 mitogen activated protein kinase.Oncogene,1999,18(51): 7253-7264
14 Yan FF,Wang B,Zhu XG,et al.Overexpression of p38MAPK protein in breast cancer and the regulation of preoperative chemotheraphy.Chin J Gen Surg,2002,17(6):343-345
15 Wang HY,Cheng Z,Mablon CC.Overexpression of mitogen-activated protein kinase phosphatases MAPK1,MAPK2 in human breast cancer.Cancer Letter.2003,191(2):229-237
16 孙泽群,徐少勇,邓长生.P38蛋白在大肠癌中的表达及其临床意义.中国肿瘤临床,2005,18(2):95-97
17 Raingeaud J,Cupta S,Rogers JS,et al.Pro-inflammatory cytokinase and envir onmental stress cause p38 mitogon - altivated protein kinase activation by dual phosphorylation on tyrosine and threonine.J BiolChem,1995,270(13):7420-7426
18 Kyriakis JM,Avruch J.Sounding the alarm:protein kinase cascades activated by stress and inflammation.J Biol Chem,1996,271(40):24313-24316
19 Tomoda K,Kubota Y,Kato J.Degradation of the cyclin-dependent kinase inhibi -tor p27kip1 is instigated by Jab1.Nature,1991,398(6723):160-165
20 Mao H,Yuan AL,Zhao MF,et al.The effect of signal passway of mitogen activated protein kinase on hepatocellular carcinoma metastasis induced by VEGF.Clin J Dig,2002,20(1):14-16
21 Greenberg AK,Basu S,Hu J,et al.Selective p38 activation in human non -small cell lung cancer.Am J Respir Cell Mol Biol,2002,26(5):558-564
1 Kin AY,Choi BY,Kim TK,et al.Hepatocellular carcinoma:power Doppler US with a contrast agent -preliminary results.Radiology,1998,209:135-140
2 袁响林,于世英,高晓会,等.人宫颈癌乏氧动物模型的建立及鉴定。肿瘤防治研究,2004,31(7):409-411
3 Braakhuis BJM.The potential of the nude mice model for the study of head and neck cancer.Arch Otolaryngol,1987,229:69-74
4 姚青,陈江浩,凌瑞,等.组织块悬液注射法制作兔VX2乳腺癌模型.中国癌症杂志,2004,14(1):22-23
5 Hanahan J,Folkman J.Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell,1996,86(3):353-364
6 Morgon J,Pake H.Metastases:mechanisms,pathways and cascades.1995,AJR,164(5):1075-1082
7 Lin WY,Chen J,Lin Y,et al.Implant5ation of VX:carcinoma into the liver of rabbits:a comparison of three direct-injection methods.Vet Med Sci,2002,64:649-652
8 王文平,徐智章,田文鹃.肝脏局灶性结节性增生的超声诊断.中华超声影像学杂志,2002,9(2):74-76
9 Iwasaki M,Furuse J,Yoshini M,et al.Sonographic appearances of small hepatic nodules without tumors on contrast-enhanced computed tomography and angiogra phy.J Clinic Ultrasound,1998,26(6):303-307
10 Furuse J,Maru Y,Yoshino M,et al.Assessnment of arteral tumor vascularity in small hepatocellular carcinoma.Comparison between color Doppler ultrasonogr -aphy,and CT hepatic arteriography.Eur J Radiol,2000,36(1):20-27
11 Strobel D,rodel U,Martus P,et al.Clinical evaluation of contrast-enhanced color Doppler sonography in the different diagnosis of liver tumors.J Clin Ultrasound,2000,28(1):1-13
12 Bude RO,Rubin IM,Adler RS,et al.Power versus conventional color Doppler sonography.Radiology,1994,192:777-780
13 Chen RC,Chen WT,Tu HY,et al.Assessment of vascularity in hepatic tumors:com -parison of power Doppler sonography and intraarterial CO(2)-enhannced power Doppler sonigraphy.AJR Am J Roentgenol,2002,178(1):67-73
14 Hasten N,Puls R,Lemke SJ,et al.Contrast-enhanced power Doppler sonography:im proved detection of characteristic flow patterns in focal liver lesions.J Clin Ultrasound,1999,27(3):107-115
15 Cedrone A,Pompili M,Sallustio G,et al.Comparison between color power duppler ultrasound with echo-enhancer and spiral computed tomography in the evaluation of hepatocellular carcinomam vascularization before and after ablation procedures.Am J Gastroenterol,2001,96(6):1854-1859
16 F.Moriyasu.Ultrasound contrast assessment of liver diseases.Preseted in 4~(th)Heart Centre European Symposium on Ultrasound Contrast Imaging,Jannary,1998,2:56-59
17 Barry B.Goldberg.Echo-enhancing agent in tumors.Advances in Echo Imaging Using Contrast Enhancement by N.C.Nanda,R.Schlief,B.B.Goldberg.Kluwer Academic Publishers,1997,18:526-531
18 Barry B.Goldberg.Role of contrast agents in ultrasonography.Applied Radiolog -y,1997,11:4-9
19 李彭,谢峰,勒宾,等.不同剂量声学造影剂对二次谐波显像的影响:剂量与效应关系.中国超声医学杂志,1998,14(12):1-3
20 刘政,王红,谢峰,等.肝脏VX2肿瘤声学造影-剂量与效应关系.中国超声医学杂志,1999,15(11):804-808
21 Tamura Y,Jimzaki M,ohkuma K,et al.Visualization of tumor vessels in renal tumors Comparison between power Doppler ultrasonography.Acta Radiol,2001,42(2):239-243
22 Rizzatto G.Towards a more sophisticated use of breast ultrasound.Eur Radiol,2001,11:2425-2435
23 钱晓芹,杨光,夏泽,等.实时超声造影成像结合多普勒血流频谱形态评价乳腺良恶性肿块的血流动力学特性.中国临床医学影像学杂志,2006,17(6):327-329
1 刘庄华,高小峰,蒋栋,等.宫颈癌的超声诊断.中国超声诊断杂志,2001,2:40-41
2 郝光荣.实验动物学(J).上海:第二军医大学出版社,1999.177-178
3 高进,李敏民.动物肿瘤模型极其应用.肿瘤学基础及实验.北京医科大学及中国协和医科大学联合出版,1992.38-87
4 杨简,高进.肿瘤.病理学(第二版).科学技术出版社,1984.784-834
5 陈小毅.全国第四界免疫缺陷动物实验研究学术交流会纪要.北京实验动物科学,1993,10(1):45-47
6 高进.动物肿瘤模型的建立极其标准研讨.中国肿瘤生物治疗杂志,1995,2(1):76-79
7 鄂征,徐新来.肿瘤细胞培养.组织培养技术(第二版).人民卫生出版社,1988,168-199
8 Sun Fx.Int J cancer.1996,66:1-5
9 Togo S.Anticancer Res,1995,15:791-794
10 孙肪宪.肿瘤模型的最新进展——“转移鼠”模型.国外医学肿瘤学分册,1996,23(5):287-289
11 Schaeffer H.J,Weber M.J.Mitogen-activated protein kinases:specific messages from ubiquitous messengers.Mol Cell Biol.1999,19:2435-2444
12 Lapor H.The α-adrenoceptor subtype mediating the tension of human prostatic smooth muscle.Prostate,1998,22:302-307
13 Kyprianou N.Apoptotic versus proliferative activities in human benign prostatic hyperplasia.Human Pathol,1996,27:668-675
14 Thieme H,Aiello LP,Takagi H,et al.Comparative analysis of vascular endothelial growth factor receptors on retinal and aortic vascular endothelial cells.Diabetes.1995,44:98-103.
15 Price D.T.Activation of extracellular signal-regulated kinase in human prostase cancer.J Urol,1999,162:1537-1542
16 Gioeli D.Activation of mitogen-prostate cancer progression.Cancer Res,1999,59:279-284
17 Davis R.J.Signal transduction by the JNK group of MAP kinase.Cell,2000,103:239-252
18 Tilton RG,Chang KC,Lejeune WS,et al.Role for nitric oxide in the hyperperme -ability and hemodynamic changes induced by intravenous VEGF.Invest Ophthalmol Vis Sci,1999 Mar,40(3):689-696.
19 Liotta LA.Cancer metastasis and angiogenesis:an imbalance of positive and negative regulation.Cell,1991,64(2):327-336
20 Merlini PA,Bauer KA,Oltrona L,et al:Persistent activation of coagulation mechanism in unstable angina and myocardial infarction.Circulation,1994,90:61-65
21 Nielsen NE,Siegbahn A,Swahn E.Marker of hypercoagulation and von willebrand factor in postmenopausal women with unstable coronary artery disease.Discriminatory ability regarding unstable coronary artery disease and coronary atherosclerlsis using receiver operating characteristics.J Intern Med,2000,248(2):151-153
22 Bang YJ,Kwon JH,Kang SH,et al.Increased MAPK activity and MKP-1 overexpression in human gastric adenocarcinoma.Biochem Biophys Res Commum,1998,250(1):43-45
23 Kikkawa F.Warny M,et al.Different activation of mitogen- activated protein kinases in AGS gastric epithelial cells by cag~+ and cag_ Helicobacter pylori.J Immunol,1999,163(10):5552-5556
24 Wang HY,Cheng Z.Overexpression of mitogenactivated protein kinase phosphatases MKP1\MKP2 in human breast cancer.Cancer Letter.2003,191(2):229-237
25 孙泽群,徐少勇,邓长生.P38蛋白在大肠癌中的表达及其临床意义.中国肿瘤临床,2005,32(1):635-637
26 Zhang XQ,Pang ZJ,Chen SL,et al.P38MAPK inhibitor SB203580 in inhibition of in vitro invision of hhuman choriocarcinoma JAR cells.J PracOncol,2003,18(2):95-97
27 Mao H,Yuan AL,Zhao MF,et al.The effect of signal passway of mitogenactivated protein kinase on hepatocellular carcinoma metastasis induced by VEGF.Clin J Dig,2002,20(1):14-16
28 Iwase T,Tanaka M,Suzuki M,et al.Identification of protein-tynosine kinase genes preferentially expressed in embryo stomach and gastric cancer.Biochem Biophys Res Common,1993,194(2):698-750
29 张桂盛.兔日本血吸虫病肝纤维化病理及超声图改变实验观察.实用寄生虫病杂志,1996;4(2):75-76
30 仇小强.145例宫颈癌病人的疗效及预后因素的COX模型分析.中国肿瘤临床,1994,21(5): 365-367;
31 Emoto M,Iwasaki H,Mimura K,et al.Differences in the angiogenesis of benign and malignant ovarian tumors,demonstrated by analyses of color Doppler ultrasou -nd,immunohistochemistry,and microvessel density.Cancer,1997,80:899-907
32 姚磊,刘军主编.医学实用数据手册.第一版,中国广播电视出版社,1993.235
33 毕素栋.超声在宫颈癌诊治中应用价值的探讨.实用医技杂志,1998,5:194-195
34 俞雯.经阴道彩色多普勒超声在妇产科应用.中国超声医学杂志,1999,15(2):144-147
35 李爱芝.宫颈癌的超声诊断.医学影像学杂志,1995,5(1):22-24。
36 Cheng WF,Wei LH.The possible use of colour flow Doppler in planning treatment in early invasive carcinoma of the cervix.Br J Obstet Gynaecol,1999,106:1137-1142
37 Cosgrove D.Why do we need contrast agents for ultrasound.Clinical Radiology,1996,51(suppl):1-5
38 Firschke C.Myocardial perfusion imaging in the setting of coronary artery setnosis and acute myocardial infarction using venous inje-ction of a second generation echocardiographic contrast agent.Circulation,1997,96:959-962
39 Wu YC,Yuan CC,Huang JH,et al.Power Doppler angiogeaphic apperance and blood flow velocity waveforms in invasive cervical carcinoma.GynecolOncol,2000,79:181-186
40 柳建华,张青萍.三维超声观察静脉注射声学造影剂前后肿瘤血流灌注的实验研究.中国超声医学杂志,1999,15(4):246-249