Midkine在胆囊癌中的表达及其意义
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摘要
背景
胆囊癌发病过程隐匿，且侵袭力强，预后较差。山于胆囊癌缺乏早期诊断的手段及其高侵袭性，胆囊癌在临床上治疗效果较差。近年来，基因研究取得了巨大进展，已发现了许多与胆囊癌密切相关的基因。随着分子生物学技术的发展，胆囊癌相关基因(如PCNA，Ki67，P16，P53，nm23等)的发现，使寻求新的胆囊癌早期诊断和治疗的方法来提高其手术切除率和生存率成为可能。
Midkine是一种新的肝素结合生长／分化因子。首先作为一个维甲酸诱导基因的产物被鉴别出来。目前已被证实的Mikdine 4种生物学功能：1．促进胚胎脑神经元的轴突向外生长。2．增加主动脉内皮细胞的血浆酶原活性。3．裸鼠体内使NIH3T3细胞癌性转化。4．促进血管生长和有丝分裂作用。除此之外，国外最新研究还表明：Mikdine对胚胎神经原和Wilm's瘤细胞具有抗凋亡作用；能促进胚胎神经原细胞、嗜中性粒细胞和巨噬细胞等各种细胞的移动；Mikdine的这两种作用可能有助于肿瘤细胞的浸润与生存。在神经母细胞瘤、神经胶质细胞瘤和膀胱癌中，高水平的Mikdine表达与临床预后有关。高分化的胃癌细胞Midkine表达明显低于低分化的胃癌细胞及印戒细胞癌。在正常成人组织中，Midkine的表达高度受限，在肾、肺、胃、结肠、卵巢和甲状腺中可检测出低水平表达的Midkine。但是在一些人的恶性肿瘤中如乳腺癌、肺癌、食管癌、胃癌、结肠癌、肝癌、卵巢癌、膀胱癌、前列腺癌、神经胶质细胞瘤、神经母细胞瘤和Wilm's瘤中观察到高水平表达的Midkine。
目前有关Mikdine在胆囊癌组织中的表达情况知之甚少，也不清楚Mikdine在胆囊癌变以及肿瘤发展过程中所扮演的角色。本实验拟通过检测Midkine蛋白在胆囊癌组织中的表达，以了解其作为胆囊肿瘤标记物的可能性，了解并研究其表达与胆囊肿瘤临床病理特征之间的关系。同时，
第二军医大学
2001级
硕L学位论文
通过比较M记kine蛋白与胆囊癌常用的增殖指标Ki67蛋白、预后指标Iun23
蛋白以及CD34表达情况，以进一步了解Midkine蛋白在胆囊癌发生发展
中扮演的角色。
目的
1、检测Midkine蛋白在胆囊癌组织及胆囊其他良性病变中的表达，探讨
M记kine蛋白的表达与胆囊癌复发、转移、年龄、性别等一般临床因
素及肿瘤直径、浸润、病理分级、分期等临床病理特征的关系。
2、初步探讨Midkine与胆囊癌血管生成、胆囊癌细胞增殖指标Ki67以及
胆囊癌转移指标nln23的关系。
方法
l、对53例胆囊癌、12例胆囊癌转移的淋巴结，12例腹腔转移灶、14例
胆囊腺瘤、20例胆囊炎性息肉、20例胆囊炎组织和18例正常胆囊病
例进行回顾，重新复习切片，明确病理诊断、病理分级。
2、以M记kine多克隆抗体免疫组化染色检测胆囊癌胆囊癌转移的淋巴
结，腹腔转移灶、胆囊腺瘤、胆囊炎性息肉、胆囊炎组织和正常胆囊
中Midkine的表达。
3、以CD34单克隆抗体、Ki一67单克隆抗体、Iun23单克隆抗体免疫组化
染色检测上述组织微血管密度(mierovessel density， Mvn)、Ki一67、
nrn23表达，并与Midkine表达比较。
4、标记指数(LI)计算标准:每一切片在低倍镜下选择组织结构良好、背
景清晰的5个阳性细胞最密集的的区域，每个区域在高倍镜视野下(X
200)计算100个细胞中的阳性细胞数(不包括间质细胞和其他非肿瘤
细胞)，取5个区域阳性细胞数的平均值作为标记指数。
5、微血管判断标准:CD34染色血管内皮细胞的胞质中出现清晰棕黄色单
个血管内皮细胞或血管细胞簇为一个血管标记，管径大于8个红细胞
直径者不列入统计之内，先在低倍镜下找到血管密度最高区域，随后在高
倍镜(x 200)下选择5个视野进行微血管数目计数，取平均值为MVD。
6、数据统i十分析方法statistieal analysis methods:Wileoxon:a砍Sum test andt
第二军医大学
2001级
硕上学位论文
check.计数及计量资料分别采用各组标记指数采用Wilcoxon秩和检验
法，检验水准取0.05，双尾检验。胆囊癌Midkine的标记指数与Ki67
的标一记指数、nm23标记指数及MVD相关系数采用t检验，检验水准取
0.05，可信区间取95%。所有数据采用SPSSg.O统计分析软件分析。
结果
1、在胆囊癌组织中，M记kine表达广泛，染色强度明显增强，染色在癌
组织内呈
弥漫分布，全部癌组织中标记指数72(41一98)。其中腺癌标记指数
70(引一91)。鳞癌标记指数73(50一94)。粘液癌中可见印戒样癌细
胞标记指数82(71一98)。
2、正常胆囊、胆囊炎组织、胆囊炎性息肉及胆囊腺瘤M记kine表达的中位
指数Ll分别为0、5(0一12)、7(0一16)与17(5一29)，与胆囊癌的MK表达的
中位指数Ll为72(41一98)比较，均有显著差异(P<0.01)。
3、胆囊癌中M记kine表达与肿瘤细胞分化程度、临床分期、有无淋巴结
转移、肝脏浸润及腹腔转移有关(P<0.05)。
4、胆囊癌组织中，新生血管、毛细血管、小静脉及小动脉内皮细胞均见
CD34表达;Ki67表达位于细胞核，胞浆未见染色，染色在癌组织内呈
弥漫分布，全部癌组织中标记指数52(35一78);，nln23表达于胞浆，细
胞核未见染色。高分化胆囊癌组织中n
Background
The procedure of gallbladder carcinoma's episode is secretiveness. Gallbladder carcinoma's invasiveness is powerful, and its prognosis is poor. Because of the deficiency of early period diagnosis method and the powful of invasiveness, gallbladder carcinoma therapeutic efficacy is very poor. In recent years, great progress has received about gene research. Many correlation genes of gallbaldder carcinoma had been found. With the progression of molecular biology, it is possible to improve gallbadder carcinoma curative effect.
Midkine is a new heparin-bingding growth/differentiation agent.lt was first distinguished as a product of gene induced by retinoic acid. Four biologic functions were confirmed at present. It promotes embryo cranial neiraxon outgrowth. It increases plasm zymogen activity of aorta endothelial cell. In nude mouse ,it makes NIH3T3 cell carcinomatous change. It promotes mitosis and angiogenesis. Oversea research also indicated that Mikdine had a role of anti-apoptosis in Wilm's tumor and embryo nerves. Midkine also facilitates transfer of embryo nerves cell, neutrophil and macrophage. These two function may help to tumor cell soakage and survive. High expression of Midkine has related to clinical prognosis in neuroblastoma, gliocytoma and bladder carcinoma. The expression of high differentiation gastric carcinoma cell is lower than low differentiation one and signet-ring cell carcinoma. In adult tissue,low expression of midkine was detected in kidney,lung,stomach,colon,ovary and thyroid gland. But hig expression
of Midkine was detected in human malignancy(breast cancer,lung cancer,esophageal carcinoma, gastric carcinoma, ovary carcinoma,bladder carcinoma,gliocytoma,neuroblastoma and Wilm's tumor.
At present,there is know few about Midkine expression in the carcinoma of gallbladder. We still don't know what is role of Midkine in the gallbladder
carcinomatous change and development. In this experiment, we will comprehend and research clinical significance of Midkine by detecting the expression of Midkine in the gallbladder carcinoma . In order to comprehend much more , we will compare the expression of midkine to nm23,Ki67and CD34 in gallbladder carcinoma.
Objective
1. To investigate the expression of Midkine in human gallbladder carcinoma and its significance in cancer biological feature.
2. To explore the relation expression of Midkine to gallbladder carcinoma's angiogenesis, cellproliferation and prognosis.
Methods
1. 53 cases of gallbladder carcinma,12 cases of metastasis lymph node, 12 cases of abdominal metastasis point ,14 cases of adenoma,20 cases of inflammatory polyp,20 cases of cholecystitis constitution and 18 cases of normal gallbladder were reviewed in this study. Expression of Midkine was evaluated by immunostaining with Midkine polyclonal antibody.
2. Various kinds of gallbladder lesion expression of Midkine was evaluated by immunostaining with Midkine polyclonal antibody.
3. Expression of Ki-67 ,nm23 were evaluated immunohistochemically with Ki-67and nm23 monoclonal antibody in gallbladder carcinma metastasis lymph node abdominal metastasis point adenoma cholecystitis constitution and normal gallbladder. Microvessel density (MVD) was examined by immunostaining with CD34 monoclonal antibody. They were compared to the expression of Midkine respectively.
4. Labelling index(LI); Microvessel density .
5. Statistical analysis methods: Wilcoxon rank sum test and t check. All of the data was analysed by SPSS9.0 software.
Results
1. Midkine is expressed in most cells of gallbladder carcinoma gallbladder carcinoma. LI of the expression of Midkine is 72 (41-98) .
2. LI of the expression of Midkine of normal gallbladder, inflammatory polyp, cholecystitis constitution, adenoma and gallbladder carcinoma is 0,5(0-12), 7(0-16) , 17(5-29) and 72(41-98). There is significant differential expression between the gallbladder carcinoma and other gallbladder constitution(P<0.01 ) .
3. There were relations between the ex
胆囊癌发病过程隐匿，且侵袭力强，预后较差。山于胆囊癌缺乏早期诊断的手段及其高侵袭性，胆囊癌在临床上治疗效果较差。近年来，基因研究取得了巨大进展，已发现了许多与胆囊癌密切相关的基因。随着分子生物学技术的发展，胆囊癌相关基因(如PCNA，Ki67，P16，P53，nm23等)的发现，使寻求新的胆囊癌早期诊断和治疗的方法来提高其手术切除率和生存率成为可能。
Midkine是一种新的肝素结合生长／分化因子。首先作为一个维甲酸诱导基因的产物被鉴别出来。目前已被证实的Mikdine 4种生物学功能：1．促进胚胎脑神经元的轴突向外生长。2．增加主动脉内皮细胞的血浆酶原活性。3．裸鼠体内使NIH3T3细胞癌性转化。4．促进血管生长和有丝分裂作用。除此之外，国外最新研究还表明：Mikdine对胚胎神经原和Wilm's瘤细胞具有抗凋亡作用；能促进胚胎神经原细胞、嗜中性粒细胞和巨噬细胞等各种细胞的移动；Mikdine的这两种作用可能有助于肿瘤细胞的浸润与生存。在神经母细胞瘤、神经胶质细胞瘤和膀胱癌中，高水平的Mikdine表达与临床预后有关。高分化的胃癌细胞Midkine表达明显低于低分化的胃癌细胞及印戒细胞癌。在正常成人组织中，Midkine的表达高度受限，在肾、肺、胃、结肠、卵巢和甲状腺中可检测出低水平表达的Midkine。但是在一些人的恶性肿瘤中如乳腺癌、肺癌、食管癌、胃癌、结肠癌、肝癌、卵巢癌、膀胱癌、前列腺癌、神经胶质细胞瘤、神经母细胞瘤和Wilm's瘤中观察到高水平表达的Midkine。
目前有关Mikdine在胆囊癌组织中的表达情况知之甚少，也不清楚Mikdine在胆囊癌变以及肿瘤发展过程中所扮演的角色。本实验拟通过检测Midkine蛋白在胆囊癌组织中的表达，以了解其作为胆囊肿瘤标记物的可能性，了解并研究其表达与胆囊肿瘤临床病理特征之间的关系。同时，
第二军医大学
2001级
硕L学位论文
通过比较M记kine蛋白与胆囊癌常用的增殖指标Ki67蛋白、预后指标Iun23
蛋白以及CD34表达情况，以进一步了解Midkine蛋白在胆囊癌发生发展
中扮演的角色。
目的
1、检测Midkine蛋白在胆囊癌组织及胆囊其他良性病变中的表达，探讨
M记kine蛋白的表达与胆囊癌复发、转移、年龄、性别等一般临床因
素及肿瘤直径、浸润、病理分级、分期等临床病理特征的关系。
2、初步探讨Midkine与胆囊癌血管生成、胆囊癌细胞增殖指标Ki67以及
胆囊癌转移指标nln23的关系。
方法
l、对53例胆囊癌、12例胆囊癌转移的淋巴结，12例腹腔转移灶、14例
胆囊腺瘤、20例胆囊炎性息肉、20例胆囊炎组织和18例正常胆囊病
例进行回顾，重新复习切片，明确病理诊断、病理分级。
2、以M记kine多克隆抗体免疫组化染色检测胆囊癌胆囊癌转移的淋巴
结，腹腔转移灶、胆囊腺瘤、胆囊炎性息肉、胆囊炎组织和正常胆囊
中Midkine的表达。
3、以CD34单克隆抗体、Ki一67单克隆抗体、Iun23单克隆抗体免疫组化
染色检测上述组织微血管密度(mierovessel density， Mvn)、Ki一67、
nrn23表达，并与Midkine表达比较。
4、标记指数(LI)计算标准:每一切片在低倍镜下选择组织结构良好、背
景清晰的5个阳性细胞最密集的的区域，每个区域在高倍镜视野下(X
200)计算100个细胞中的阳性细胞数(不包括间质细胞和其他非肿瘤
细胞)，取5个区域阳性细胞数的平均值作为标记指数。
5、微血管判断标准:CD34染色血管内皮细胞的胞质中出现清晰棕黄色单
个血管内皮细胞或血管细胞簇为一个血管标记，管径大于8个红细胞
直径者不列入统计之内，先在低倍镜下找到血管密度最高区域，随后在高
倍镜(x 200)下选择5个视野进行微血管数目计数，取平均值为MVD。
6、数据统i十分析方法statistieal analysis methods:Wileoxon:a砍Sum test andt
第二军医大学
2001级
硕上学位论文
check.计数及计量资料分别采用各组标记指数采用Wilcoxon秩和检验
法，检验水准取0.05，双尾检验。胆囊癌Midkine的标记指数与Ki67
的标一记指数、nm23标记指数及MVD相关系数采用t检验，检验水准取
0.05，可信区间取95%。所有数据采用SPSSg.O统计分析软件分析。
结果
1、在胆囊癌组织中，M记kine表达广泛，染色强度明显增强，染色在癌
组织内呈
弥漫分布，全部癌组织中标记指数72(41一98)。其中腺癌标记指数
70(引一91)。鳞癌标记指数73(50一94)。粘液癌中可见印戒样癌细
胞标记指数82(71一98)。
2、正常胆囊、胆囊炎组织、胆囊炎性息肉及胆囊腺瘤M记kine表达的中位
指数Ll分别为0、5(0一12)、7(0一16)与17(5一29)，与胆囊癌的MK表达的
中位指数Ll为72(41一98)比较，均有显著差异(P<0.01)。
3、胆囊癌中M记kine表达与肿瘤细胞分化程度、临床分期、有无淋巴结
转移、肝脏浸润及腹腔转移有关(P<0.05)。
4、胆囊癌组织中，新生血管、毛细血管、小静脉及小动脉内皮细胞均见
CD34表达;Ki67表达位于细胞核，胞浆未见染色，染色在癌组织内呈
弥漫分布，全部癌组织中标记指数52(35一78);，nln23表达于胞浆，细
胞核未见染色。高分化胆囊癌组织中n
Background
The procedure of gallbladder carcinoma's episode is secretiveness. Gallbladder carcinoma's invasiveness is powerful, and its prognosis is poor. Because of the deficiency of early period diagnosis method and the powful of invasiveness, gallbladder carcinoma therapeutic efficacy is very poor. In recent years, great progress has received about gene research. Many correlation genes of gallbaldder carcinoma had been found. With the progression of molecular biology, it is possible to improve gallbadder carcinoma curative effect.
Midkine is a new heparin-bingding growth/differentiation agent.lt was first distinguished as a product of gene induced by retinoic acid. Four biologic functions were confirmed at present. It promotes embryo cranial neiraxon outgrowth. It increases plasm zymogen activity of aorta endothelial cell. In nude mouse ,it makes NIH3T3 cell carcinomatous change. It promotes mitosis and angiogenesis. Oversea research also indicated that Mikdine had a role of anti-apoptosis in Wilm's tumor and embryo nerves. Midkine also facilitates transfer of embryo nerves cell, neutrophil and macrophage. These two function may help to tumor cell soakage and survive. High expression of Midkine has related to clinical prognosis in neuroblastoma, gliocytoma and bladder carcinoma. The expression of high differentiation gastric carcinoma cell is lower than low differentiation one and signet-ring cell carcinoma. In adult tissue,low expression of midkine was detected in kidney,lung,stomach,colon,ovary and thyroid gland. But hig expression
of Midkine was detected in human malignancy(breast cancer,lung cancer,esophageal carcinoma, gastric carcinoma, ovary carcinoma,bladder carcinoma,gliocytoma,neuroblastoma and Wilm's tumor.
At present,there is know few about Midkine expression in the carcinoma of gallbladder. We still don't know what is role of Midkine in the gallbladder
carcinomatous change and development. In this experiment, we will comprehend and research clinical significance of Midkine by detecting the expression of Midkine in the gallbladder carcinoma . In order to comprehend much more , we will compare the expression of midkine to nm23,Ki67and CD34 in gallbladder carcinoma.
Objective
1. To investigate the expression of Midkine in human gallbladder carcinoma and its significance in cancer biological feature.
2. To explore the relation expression of Midkine to gallbladder carcinoma's angiogenesis, cellproliferation and prognosis.
Methods
1. 53 cases of gallbladder carcinma,12 cases of metastasis lymph node, 12 cases of abdominal metastasis point ,14 cases of adenoma,20 cases of inflammatory polyp,20 cases of cholecystitis constitution and 18 cases of normal gallbladder were reviewed in this study. Expression of Midkine was evaluated by immunostaining with Midkine polyclonal antibody.
2. Various kinds of gallbladder lesion expression of Midkine was evaluated by immunostaining with Midkine polyclonal antibody.
3. Expression of Ki-67 ,nm23 were evaluated immunohistochemically with Ki-67and nm23 monoclonal antibody in gallbladder carcinma metastasis lymph node abdominal metastasis point adenoma cholecystitis constitution and normal gallbladder. Microvessel density (MVD) was examined by immunostaining with CD34 monoclonal antibody. They were compared to the expression of Midkine respectively.
4. Labelling index(LI); Microvessel density .
5. Statistical analysis methods: Wilcoxon rank sum test and t check. All of the data was analysed by SPSS9.0 software.
Results
1. Midkine is expressed in most cells of gallbladder carcinoma gallbladder carcinoma. LI of the expression of Midkine is 72 (41-98) .
2. LI of the expression of Midkine of normal gallbladder, inflammatory polyp, cholecystitis constitution, adenoma and gallbladder carcinoma is 0,5(0-12), 7(0-16) , 17(5-29) and 72(41-98). There is significant differential expression between the gallbladder carcinoma and other gallbladder constitution(P<0.01 ) .
3. There were relations between the ex
引文
1. Cubertafond P, et al. Surgical treatment of the gallbladder.Ann surg.1994; 219: 322-334.
2. Tomomura M, Muramatasu T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 1988; 151:1312-1318.
3. Tomomura M, Kadomatsu K, Matsubara S, et al. A retinoic acid-responsive gene, Mikdine, found in the terato carcinoma system. Heterogeneity of the transcript and the nature of the translation product. J Biol Chem 1990;265:10765-10770.
4. Kaname T, Kuwano A, Murano I, et al. Midkine gene, a gene for prenatal differentiation and neuroregulation. Maps to band 11p11.2 by fluorescence in situ-hybridization. Genomics 1993; 17:514-515.
5. Uehara K, Matsubara S, Kadomatsu K, et al. Genomic structure of human midkine (Mikdine), a retinoic acid-responsive growth/differentiation factor. J
Biochem Tokyo 1992;111: 563-567.
6. Nakanishi T, Kadomatsu K, Okamoto T, et al. Expression of midkine and pleiotropin in ovarian tumors. Obstet Gynecol 1997;90:285-290.
7. Choudhuri R, Zhang HT, Donnini S, et al. An angiogenic role for the neurokines midkine and pleiotrophin in tumorigenesis. Cancer Res 1997;57:1814-1819.
8. Qi M., IkematsuS., Ichihara-Tanaka, K., Sakuma, S., Muramatsu,T., and Kadomatsu, K. Midkine rescues Wilms' tumor cells from cisplatin-induced apoptosis: regulation of Bcl-2 expression by midkine. J. Biochem. 2000; 127: 269 - 277.
9. Waichi S, Kenji K, Yukio Y, et al. Midkine Is Involved in Neutrophil Infiltration into the Tubulointerstitium in Ischemic Renal Injury J. Immu. 2001;167: 3463-3469.
10. kematsu S, Yano A, Aridome K, et al. Serum midkine levels are increased in patients with various types of carcinomas. Br J Cancer 2000;83:701-706.
11. Nakanishi T, Kadomatsu K, Okamoto T, et al. Expression of midkine and pleiotropin in ovarian tumors. Obstet Gynecol 1997;90:285-290
12. Tsutsui J, Kadomatsu K, Matsubara S, et al. A new family of heparin- binding growth/differentiation factors: increased midkine expression in Wilms' tumor and other human carcinomas. Cancer Res 1993;53:1281-1285.
13. O'Bnein T, Cranston Denggle Set al.The angiogenic factor midkine is expressed in and overexpression correlates with a poor outcome in patients with invasive cancer. Cancer Res. 1996;56:2515-2518
14. Schluter C,Duchrow M,Wohlenberg C et al.The cell proliferation-associated nuclear antigen of antibody Ki67:A very large,ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J cell biol. 1993;123:513-521
15. Sawan A,LascuI,Veron M,et al.NDPK/nm23 expression in human breast cancer in relation to relapse, survival, and ther prognostic factors:An immunohitochemical study. J Pathol,1994;172:27-39.
16．CD34基因与血管内皮细胞 罗杰 王长谦 综述 国外医学．生理、病理科学与
临床分册2000；6：442-444。
17. Acenero MJ,Gonzalez JF, Gallego MG, et al.Vascular enumerationation as a significant prognosticator for invasive breast carcinoma.J Clinical Oncology.1998; 16:1684-1688.
18. Ohama H, Malsubara S, Guenet J. et al. The midkine family of growth/differentiation factors; structure of a Mikdine-relationship among member of the Mikdine family.Jbiochem,1994;115:516-522
19. Satyamoorthy, K., Oka, M., and Herlyn, M An antisense strategy for inhibition of human melanoma growth targets the growth factor pleiotrophin. Pigment Cell Res.2000; 13 Supple 8:87 - 93
20. 19.Muramatsu, H., Inui, T., Kimura, T., Sakakibara, S., et al. Localization of heparinbinding,neurite outgrowth and antigenic regions in midkine molecule. Biochem. Biophys. Res. Commun. 1994;203:1131-1139
21. 20Akhter, S., Ichihara-Tanaka, K., Kojima, S., Muramatsu, H.,Inui, T., Kimura, T., Kaneda, N., Talukder, A.H., Kadomatsu,K., Inagaki, F., and Muramatsu, T. Clusters of basic amino acids in midkine: roles in neurite-promoting activity and plasminogen activator-enhancing activity. J. Biochem. 1998; 123:1127 - 1136
22. Fu C,Maminta-Smith L,Guo C et al.Cloning and sequence of the xenopus laevis homologue of the midkine cDNA.Gene. 1994; 146:311-312
23. Michikawa, M., Kikuchi, S., Muramatsu, H., Muramatsu, T., and Kim, S.U. Retinoic acid responsive gene product,Midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture.J. Neurosci.Res. 1993;35:530 - 539
24. Satoh, J., Muramatsu, H., Moretto, G., Muramatsu, T., Chang,H.J., Kim, S.T., Cho, J.M., and Kim, S.U. Midkine that promotes survival of human neurons is produced by fetal human astrocytes in culture. Dev. Brain Res. 1993;75:201 - 205
25. Kikuchi, S., Muramatsu, H., Muramatsu, T., and Kim, S.U. Midkine, a novel neurotrophic factor, promotes survival of mesencephalic neurons in culture. Neurosci Lett. 1993;160: 9-12
26. Adachs Y, Mausubara S,Pedraza C et al.Midkine as a novel target gene for the
Wilm's tumor suppressor gene.Oncogen, 1996; 113:2197-2203
27. Aridome,-K; Tsutsui,-J; Takao,-S; Kadomatsu,-K; Ozawa,-M; Aikou,-T; Muramatsu. Increased midkine gene expression in human gastrointestinal cancers. Jpn J. Cancer Res. 1995; 86:655-661
28. Aridome K,Takao S,Kaname Tetal. Truncated midkine as a marker diagnosis and detection of node metastasis on gastrointestinal carcinmas. Br J Cancer. 1998;78:472-477
29. Miyashiro I,Kaname Tshin E et al. Expression of truncated midkine in human colonectal cancers. Breast cancer Res Treat. 1997;43:1-6
30. Ratoviski EA, Burrow CR, Midkine stimulates wilm's cell proliferation via its signaling receptor.Cell Mol Biol. 1997;43:425-431
31. Gaver RI, Radford DM,Donis-keller H et al.Midkine and pleiotrophin expression in normal and malignant breast tissues.Cancer, 1995;74:1584-1590
32. Muramatasu H, Song Xj.Koide N et al.Enzyme-linked immunoassay for midkine and its application to evaluation of midkine levels in developing mouse brain sera from patients with hepatocellular carcinomas. J Biochem. 1996; 199:1171-1175
33. Ye, CAkiyama, S., Kasai, Y., Matsuyama,M., Muramatsu, T., and Kadomatsu, K. Expression of midkine in the early stage of carcinogenesis in human colorectal cancer. Brit. J. Cancer. 1999; 79:179-184
34. Sakitani, H., Tsutsumi, M., Kadomatsu, K., Ikematsu, S., Takahama, M. et al. Overexpression of midkine in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine in rats and its increase with progression. Carcinogenesis 1999;20:465 - 469
35. Zhang N;Deuel T;et al. Pleiotrophin and midkine, a family of mitogenic and angiogenic heparin-binding growth and differentiation factors. Curr-Opin-Hematol. 1999 Jan; 6(1): 44-50
36．郭学良 傅丽娜 Ki67在大肠肿瘤研究中的应用 复旦学报(科学医学版)2001；July，28：372-374。
37. John G. Wesseling, Masato Yamamoto,et al. Midkine and cyclooxygenase-2 promoters are promising for adenoviral vector gene delivery of pancreatic
carcinoma Cancer Gene Therapy 2001: 8; 990-996.
38. Qi M., IkematsuS., Ichihara-Tanaka, K., Sakuma, S., Muramatsu,T., and Kadomatsu, K. Midkine rescues Wilms' tumor cells from cisplatin-induced apoptosis: regulation of Bcl-2 expression by midkine. J. Biochem. 2000; 127: 269-277
2. Tomomura M, Muramatasu T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 1988; 151:1312-1318.
3. Tomomura M, Kadomatsu K, Matsubara S, et al. A retinoic acid-responsive gene, Mikdine, found in the terato carcinoma system. Heterogeneity of the transcript and the nature of the translation product. J Biol Chem 1990;265:10765-10770.
4. Kaname T, Kuwano A, Murano I, et al. Midkine gene, a gene for prenatal differentiation and neuroregulation. Maps to band 11p11.2 by fluorescence in situ-hybridization. Genomics 1993; 17:514-515.
5. Uehara K, Matsubara S, Kadomatsu K, et al. Genomic structure of human midkine (Mikdine), a retinoic acid-responsive growth/differentiation factor. J
Biochem Tokyo 1992;111: 563-567.
6. Nakanishi T, Kadomatsu K, Okamoto T, et al. Expression of midkine and pleiotropin in ovarian tumors. Obstet Gynecol 1997;90:285-290.
7. Choudhuri R, Zhang HT, Donnini S, et al. An angiogenic role for the neurokines midkine and pleiotrophin in tumorigenesis. Cancer Res 1997;57:1814-1819.
8. Qi M., IkematsuS., Ichihara-Tanaka, K., Sakuma, S., Muramatsu,T., and Kadomatsu, K. Midkine rescues Wilms' tumor cells from cisplatin-induced apoptosis: regulation of Bcl-2 expression by midkine. J. Biochem. 2000; 127: 269 - 277.
9. Waichi S, Kenji K, Yukio Y, et al. Midkine Is Involved in Neutrophil Infiltration into the Tubulointerstitium in Ischemic Renal Injury J. Immu. 2001;167: 3463-3469.
10. kematsu S, Yano A, Aridome K, et al. Serum midkine levels are increased in patients with various types of carcinomas. Br J Cancer 2000;83:701-706.
11. Nakanishi T, Kadomatsu K, Okamoto T, et al. Expression of midkine and pleiotropin in ovarian tumors. Obstet Gynecol 1997;90:285-290
12. Tsutsui J, Kadomatsu K, Matsubara S, et al. A new family of heparin- binding growth/differentiation factors: increased midkine expression in Wilms' tumor and other human carcinomas. Cancer Res 1993;53:1281-1285.
13. O'Bnein T, Cranston Denggle Set al.The angiogenic factor midkine is expressed in and overexpression correlates with a poor outcome in patients with invasive cancer. Cancer Res. 1996;56:2515-2518
14. Schluter C,Duchrow M,Wohlenberg C et al.The cell proliferation-associated nuclear antigen of antibody Ki67:A very large,ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J cell biol. 1993;123:513-521
15. Sawan A,LascuI,Veron M,et al.NDPK/nm23 expression in human breast cancer in relation to relapse, survival, and ther prognostic factors:An immunohitochemical study. J Pathol,1994;172:27-39.
16．CD34基因与血管内皮细胞 罗杰 王长谦 综述 国外医学．生理、病理科学与
临床分册2000；6：442-444。
17. Acenero MJ,Gonzalez JF, Gallego MG, et al.Vascular enumerationation as a significant prognosticator for invasive breast carcinoma.J Clinical Oncology.1998; 16:1684-1688.
18. Ohama H, Malsubara S, Guenet J. et al. The midkine family of growth/differentiation factors; structure of a Mikdine-relationship among member of the Mikdine family.Jbiochem,1994;115:516-522
19. Satyamoorthy, K., Oka, M., and Herlyn, M An antisense strategy for inhibition of human melanoma growth targets the growth factor pleiotrophin. Pigment Cell Res.2000; 13 Supple 8:87 - 93
20. 19.Muramatsu, H., Inui, T., Kimura, T., Sakakibara, S., et al. Localization of heparinbinding,neurite outgrowth and antigenic regions in midkine molecule. Biochem. Biophys. Res. Commun. 1994;203:1131-1139
21. 20Akhter, S., Ichihara-Tanaka, K., Kojima, S., Muramatsu, H.,Inui, T., Kimura, T., Kaneda, N., Talukder, A.H., Kadomatsu,K., Inagaki, F., and Muramatsu, T. Clusters of basic amino acids in midkine: roles in neurite-promoting activity and plasminogen activator-enhancing activity. J. Biochem. 1998; 123:1127 - 1136
22. Fu C,Maminta-Smith L,Guo C et al.Cloning and sequence of the xenopus laevis homologue of the midkine cDNA.Gene. 1994; 146:311-312
23. Michikawa, M., Kikuchi, S., Muramatsu, H., Muramatsu, T., and Kim, S.U. Retinoic acid responsive gene product,Midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture.J. Neurosci.Res. 1993;35:530 - 539
24. Satoh, J., Muramatsu, H., Moretto, G., Muramatsu, T., Chang,H.J., Kim, S.T., Cho, J.M., and Kim, S.U. Midkine that promotes survival of human neurons is produced by fetal human astrocytes in culture. Dev. Brain Res. 1993;75:201 - 205
25. Kikuchi, S., Muramatsu, H., Muramatsu, T., and Kim, S.U. Midkine, a novel neurotrophic factor, promotes survival of mesencephalic neurons in culture. Neurosci Lett. 1993;160: 9-12
26. Adachs Y, Mausubara S,Pedraza C et al.Midkine as a novel target gene for the
Wilm's tumor suppressor gene.Oncogen, 1996; 113:2197-2203
27. Aridome,-K; Tsutsui,-J; Takao,-S; Kadomatsu,-K; Ozawa,-M; Aikou,-T; Muramatsu. Increased midkine gene expression in human gastrointestinal cancers. Jpn J. Cancer Res. 1995; 86:655-661
28. Aridome K,Takao S,Kaname Tetal. Truncated midkine as a marker diagnosis and detection of node metastasis on gastrointestinal carcinmas. Br J Cancer. 1998;78:472-477
29. Miyashiro I,Kaname Tshin E et al. Expression of truncated midkine in human colonectal cancers. Breast cancer Res Treat. 1997;43:1-6
30. Ratoviski EA, Burrow CR, Midkine stimulates wilm's cell proliferation via its signaling receptor.Cell Mol Biol. 1997;43:425-431
31. Gaver RI, Radford DM,Donis-keller H et al.Midkine and pleiotrophin expression in normal and malignant breast tissues.Cancer, 1995;74:1584-1590
32. Muramatasu H, Song Xj.Koide N et al.Enzyme-linked immunoassay for midkine and its application to evaluation of midkine levels in developing mouse brain sera from patients with hepatocellular carcinomas. J Biochem. 1996; 199:1171-1175
33. Ye, CAkiyama, S., Kasai, Y., Matsuyama,M., Muramatsu, T., and Kadomatsu, K. Expression of midkine in the early stage of carcinogenesis in human colorectal cancer. Brit. J. Cancer. 1999; 79:179-184
34. Sakitani, H., Tsutsumi, M., Kadomatsu, K., Ikematsu, S., Takahama, M. et al. Overexpression of midkine in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine in rats and its increase with progression. Carcinogenesis 1999;20:465 - 469
35. Zhang N;Deuel T;et al. Pleiotrophin and midkine, a family of mitogenic and angiogenic heparin-binding growth and differentiation factors. Curr-Opin-Hematol. 1999 Jan; 6(1): 44-50
36．郭学良 傅丽娜 Ki67在大肠肿瘤研究中的应用 复旦学报(科学医学版)2001；July，28：372-374。
37. John G. Wesseling, Masato Yamamoto,et al. Midkine and cyclooxygenase-2 promoters are promising for adenoviral vector gene delivery of pancreatic
carcinoma Cancer Gene Therapy 2001: 8; 990-996.
38. Qi M., IkematsuS., Ichihara-Tanaka, K., Sakuma, S., Muramatsu,T., and Kadomatsu, K. Midkine rescues Wilms' tumor cells from cisplatin-induced apoptosis: regulation of Bcl-2 expression by midkine. J. Biochem. 2000; 127: 269-277