KAI1基因对肝癌MHCC97-H细胞生长及侵袭能力的影响
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摘要
目的：KAI1基因是新近发现的肿瘤转移抑制基因。本课题试图通过基因转染技术，探讨KAI1基因对肝癌MHCC97-H细胞生长及侵袭能力的影响，为肝癌后续的抗转移治疗研究奠定基础。
方法：将利用亚克隆技术构建的人类KAI1全长正、反义结构基因哺乳动物真核表达质粒，通过DOTAP脂质体介导的转染技术分别转入人肝癌MHCC97-H细胞中，经聚合酶链反应（PCR）对转染基因的整合鉴定和Western blot分析的表达鉴定后，观察基因转染对细胞超微结构、KAI1蛋白表达、体外生长状况、细胞周期、集落形成能力以及体外侵袭能力等的影响。
结果：(1) 电镜观察基因转染前后的细胞在大体形态、表面微绒毛、核型及分裂像等方面均未见到明显变化；但转染正义KAI1基因后细胞线粒体数量较对照组减少，转染反义KAI1基因后则细胞线粒体数量增多、体积增大，并可见髓鞘样变，粗面内质网、Golgi较发达，内质网扩张。 (2)细胞体外生长曲线及细胞周期：连续观察10天，正、反义KAI1基因转染组与对照组MHCC97-H在细胞生长曲线方面未见明显差异；用流式细胞仪分析细胞周期也无明显差别。(3)Western blot 分析和免疫细胞化学SP法染色显示，MHCC97-H细胞在转染正义基因后KAI1蛋白表达增强，相反，转染反义基因后KAI1蛋白表达减弱。KAI1蛋白主要表达于细胞浆。（4）平板培养2周后，细胞集落形成数与对照组MHCC97-H（38.50±1.91）比较，转染KAI1正义基因后（36.25±3.30）下降不显著（P>0.05），而转染KAI1反义基因后（132.50±12.87）则显著增多（P<0.01）。（5）体外侵袭实验发现，转染KAI1正义基因后的穿膜细胞数（59.67±3.51）较对照组MHCC97-H细胞（92.67±1.53）明显减少（P<0.01），而转染KAI1反义基因后的穿膜细胞数（188.00±4.51）则较对照组明显增加（P<0.01）。
结论：（1）构建了KAI1正、反义基因哺乳动物真核表达质粒，并分别成功转染给高转移潜能的人肝癌MHCC97-H细胞。（2）首次发现转染KAI1基因可导致肝癌MHCC97-H细胞一些细胞器发生数量和形态学方面的变化。（3）KAI1基因对肝癌MHCC97-H细胞的体外生长曲线及细胞周期无明显影响。（4）反义KAI1基因可使MHCC97-H细胞的克隆形成能力增强，但正义KAI1基因使MHCC97-H细胞的克隆形成能力下降的作用则不显著。（5）KAI1基因上调表达能使MHCC97-H细胞的侵袭能力降低。提示上调KAI1基因表达可能是肝癌抗转移研究的一个方向。
Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. Metastasis and recurrence are the most principal factors for their prognosis of the patients with the tumor. KAI1 gene, isolated from human metastatic prostate tumor in 1995, was regarded as a metastasis suppressor gene for prostate cancer. Further studies showed decreased KAI1 expression was observed not only in human prostate cancer but also in several common solid epithelial tumors. The aim of present study is to explore the effects of KAI1 gene on the growth and invasion of HCC cell line MHCC97-H.
Methods: KAI1 sense and antisense eukaryotic expression plasmids were constructed using subclone technique and transfected into MHCC97-H cells respectively by DOTAP liposome transfection system to directly interfere in the expression of KAI1 gene. PCR was used to confirm the transfection, and Western blot to understand if the recombinants could function in cells. Several items about cell growth and invasion were measured.
Results: (1) Two recombinants from subclone construction were digested and resulted in two fragments respectively as 0.75kb and 7.72kb, 2.25kb and 6.22kb. These results were consistent with expected recombinant plasmids. (2) PCR showed a specific fragment with length 790bp was amplified from MHCC97-H-S cells (transfected with sense KAI1), MHCC97-H-AS (transfected with antisense KAI1) and MHCC97-H-pCI (transfected with vector pCI-neo), but none from MHCC97-H. The results exhibited that the recombinants and the vector pCI-neo had integrated into the chromosome of MHCC97-H cells respectively. Western blot analysis showed enhanced expression of KAI1 protein in MHCC97-H-S, but decreased expression in MHCC97-H-AS, and no obvious difference in MHCC97-H-pCI in contrast with MHCC97-H. (3) No significant differences in growth curve and cell cycles were observed among MHCC97-H-S, MHCC97-H-AS, and MHCC97-H. (4) Under observation with electrical microscope, the amount of mitochondrions was decreased in MHCC97-H-S cells, but increased in MHCC97-H-AS cells. Furthermore, increased amount of rough endoplasmic reticulum, Golgi apparatus and
expanded endoplasmic reticulum was also noted in MHCC97-H-AS cells. (5) KAI1 immunoreaction was exhibited in the cell cytoplasm. Stronger staining was present in MHCC97-H-S whereas weaker staining in MHCC97-H-AS compared with MHCC97-H. (6)Plate cloning test showed there existed no statistical difference between MHCC97-H-S（36.25±3.30） and MHCC97-H （38.50±1.91）(P>0.05). However, MHCC97-H-AS （132.50±12.87）showed higher clone formation (P<0.01). (7) Boyden Chamber test revealed that the cells penetrating the artificial basement membrane in MHCC97-H-S（59.67±3.51） were fewer than that in MHCC97-H（92.67±1.53） (P<0.01), however, more penetrating cells in MHCC97-H-AS （188.00±4.51）were noted as compared with that in MHCC97-H (P<0.01).
Conclusion: Sense and antisense KAI1 eukaryotic expression plasmids were successfully constructed and transfected into MHCC97-H respectively. Sense KAI1 gene could upregulate the expression of KAI1 protein in MHCC97-H cells, however, antisense KAI1 gene could downregulate the expression of KAI1 protein. KAI1 gene had no obvious effects on cell cycles and growth of MHCC97-H, but could alter amount and morphology of some organells. Enhanced KAI1 expression could decrease the invasive ability of MHCC97-H. These results suggest that it may be an effective route to upregulate KAI1 expression for inhibiting the metastasis of HCC.
方法：将利用亚克隆技术构建的人类KAI1全长正、反义结构基因哺乳动物真核表达质粒，通过DOTAP脂质体介导的转染技术分别转入人肝癌MHCC97-H细胞中，经聚合酶链反应（PCR）对转染基因的整合鉴定和Western blot分析的表达鉴定后，观察基因转染对细胞超微结构、KAI1蛋白表达、体外生长状况、细胞周期、集落形成能力以及体外侵袭能力等的影响。
结果：(1) 电镜观察基因转染前后的细胞在大体形态、表面微绒毛、核型及分裂像等方面均未见到明显变化；但转染正义KAI1基因后细胞线粒体数量较对照组减少，转染反义KAI1基因后则细胞线粒体数量增多、体积增大，并可见髓鞘样变，粗面内质网、Golgi较发达，内质网扩张。 (2)细胞体外生长曲线及细胞周期：连续观察10天，正、反义KAI1基因转染组与对照组MHCC97-H在细胞生长曲线方面未见明显差异；用流式细胞仪分析细胞周期也无明显差别。(3)Western blot 分析和免疫细胞化学SP法染色显示，MHCC97-H细胞在转染正义基因后KAI1蛋白表达增强，相反，转染反义基因后KAI1蛋白表达减弱。KAI1蛋白主要表达于细胞浆。（4）平板培养2周后，细胞集落形成数与对照组MHCC97-H（38.50±1.91）比较，转染KAI1正义基因后（36.25±3.30）下降不显著（P>0.05），而转染KAI1反义基因后（132.50±12.87）则显著增多（P<0.01）。（5）体外侵袭实验发现，转染KAI1正义基因后的穿膜细胞数（59.67±3.51）较对照组MHCC97-H细胞（92.67±1.53）明显减少（P<0.01），而转染KAI1反义基因后的穿膜细胞数（188.00±4.51）则较对照组明显增加（P<0.01）。
结论：（1）构建了KAI1正、反义基因哺乳动物真核表达质粒，并分别成功转染给高转移潜能的人肝癌MHCC97-H细胞。（2）首次发现转染KAI1基因可导致肝癌MHCC97-H细胞一些细胞器发生数量和形态学方面的变化。（3）KAI1基因对肝癌MHCC97-H细胞的体外生长曲线及细胞周期无明显影响。（4）反义KAI1基因可使MHCC97-H细胞的克隆形成能力增强，但正义KAI1基因使MHCC97-H细胞的克隆形成能力下降的作用则不显著。（5）KAI1基因上调表达能使MHCC97-H细胞的侵袭能力降低。提示上调KAI1基因表达可能是肝癌抗转移研究的一个方向。
Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. Metastasis and recurrence are the most principal factors for their prognosis of the patients with the tumor. KAI1 gene, isolated from human metastatic prostate tumor in 1995, was regarded as a metastasis suppressor gene for prostate cancer. Further studies showed decreased KAI1 expression was observed not only in human prostate cancer but also in several common solid epithelial tumors. The aim of present study is to explore the effects of KAI1 gene on the growth and invasion of HCC cell line MHCC97-H.
Methods: KAI1 sense and antisense eukaryotic expression plasmids were constructed using subclone technique and transfected into MHCC97-H cells respectively by DOTAP liposome transfection system to directly interfere in the expression of KAI1 gene. PCR was used to confirm the transfection, and Western blot to understand if the recombinants could function in cells. Several items about cell growth and invasion were measured.
Results: (1) Two recombinants from subclone construction were digested and resulted in two fragments respectively as 0.75kb and 7.72kb, 2.25kb and 6.22kb. These results were consistent with expected recombinant plasmids. (2) PCR showed a specific fragment with length 790bp was amplified from MHCC97-H-S cells (transfected with sense KAI1), MHCC97-H-AS (transfected with antisense KAI1) and MHCC97-H-pCI (transfected with vector pCI-neo), but none from MHCC97-H. The results exhibited that the recombinants and the vector pCI-neo had integrated into the chromosome of MHCC97-H cells respectively. Western blot analysis showed enhanced expression of KAI1 protein in MHCC97-H-S, but decreased expression in MHCC97-H-AS, and no obvious difference in MHCC97-H-pCI in contrast with MHCC97-H. (3) No significant differences in growth curve and cell cycles were observed among MHCC97-H-S, MHCC97-H-AS, and MHCC97-H. (4) Under observation with electrical microscope, the amount of mitochondrions was decreased in MHCC97-H-S cells, but increased in MHCC97-H-AS cells. Furthermore, increased amount of rough endoplasmic reticulum, Golgi apparatus and
expanded endoplasmic reticulum was also noted in MHCC97-H-AS cells. (5) KAI1 immunoreaction was exhibited in the cell cytoplasm. Stronger staining was present in MHCC97-H-S whereas weaker staining in MHCC97-H-AS compared with MHCC97-H. (6)Plate cloning test showed there existed no statistical difference between MHCC97-H-S（36.25±3.30） and MHCC97-H （38.50±1.91）(P>0.05). However, MHCC97-H-AS （132.50±12.87）showed higher clone formation (P<0.01). (7) Boyden Chamber test revealed that the cells penetrating the artificial basement membrane in MHCC97-H-S（59.67±3.51） were fewer than that in MHCC97-H（92.67±1.53） (P<0.01), however, more penetrating cells in MHCC97-H-AS （188.00±4.51）were noted as compared with that in MHCC97-H (P<0.01).
Conclusion: Sense and antisense KAI1 eukaryotic expression plasmids were successfully constructed and transfected into MHCC97-H respectively. Sense KAI1 gene could upregulate the expression of KAI1 protein in MHCC97-H cells, however, antisense KAI1 gene could downregulate the expression of KAI1 protein. KAI1 gene had no obvious effects on cell cycles and growth of MHCC97-H, but could alter amount and morphology of some organells. Enhanced KAI1 expression could decrease the invasive ability of MHCC97-H. These results suggest that it may be an effective route to upregulate KAI1 expression for inhibiting the metastasis of HCC.
引文
1. 李雁,汤钊猷,叶胜龙等.体内连续筛选法建立自发性肺转移人肝癌细胞系.中华医学杂志.2002; 82: 601-605.
2. J·萨姆布鲁克,E.F.弗里奇,T·曼尼阿蒂斯著.金东雁,黎孟枫译.分子克隆实验指南.科学技术出版社出版,北京,第二版.1998.
3. 姜泊,张亚历,周殿元主编.分子生物学常用实验方法.人民军医出版社.北京.第一版.1996.
4. 张腾飞,陈诗书.γ-干拢素基因的导入及删除对人肿瘤细胞HLA-I、II类抗原表达的影响.胃肠病学1999,1:6-7.
5. F.奥斯伯,R.E.金斯顿,J.G.塞德曼等著.颜子颖,王海林译.精编分子生物学实验指南. 科学技术出版社,北京,第一版,1999.
6. 司徒镇强,吴军正主编.细胞培养.世界图书出版公司.西安.第一版.1996.
7. 朱金海,黄建富,陈燕凌等.PCNA反义寡核苷酸抑制人肝癌细胞体外增殖的研究.肝胆外科杂志.2002;10:65-68.
8. 杜月光,陈慰浙,陈智.MMP-7反义寡核苷酸对KATO Ⅲ细胞MMP-7 mRNA表达及其侵袭力的影响.中国病理生理杂志.2002,18:172-175
9. 傅建民, 余小舫, 邵永孚. 端粒酶与原发性肝癌. 世界华人消化杂志.2000; 8: 461-463.
10. 翟守恒, 刘俊彬, 刘云梅, 等.HBsAg, HCV-Ag和AFP在肝硬变和肝细胞癌组织中的表达. 世界华人消化杂志.2000;8:524-527.
11. Yang JM(杨建民),Chen WS(陈文生),Liu ZP(刘志鹏),et al.Effects of insulin-like growth factors-IR and -ⅡR antisense gene transfection on the biological behaviors of SMMC-7721 human hepatoma cells.J Gastroenterol Hepatol, 2003;18:296-301.
12. 吴孟超.中国原发性肝癌的临床和实验研究.中德临床肿瘤杂志.2002,1:1-4.
13. Zamanaka N,Okamoto E,Toyosaka A,et al.Prognostic factors after hepatectomy for hepatocellular carcinomas.Cancer 1990;65:1104-1110.
14. Shirabe K,Kanematsu T,Matsumata T,et al. Factors linked to early recurrence of small hepatocellular carcinoma after hepatectomy: univariate and multivariate analyses. Hepatology 1991; 14: 802-805.
Ikeda k,Saitou S,Tsubota A,et al.Risk factors for tumor recurrence and prognosis after
15. curative resection of hepatocellular carcinoma.Cancer 1993;71:19-25.
16. Arii S,Tanaka J,Yamazoe Y,et al.Predictive factors for intrahepatic recurrence of hepatocellular carcinoma after partial hepatectomy.Cancer 1992;69:913-919.
17. Mashimo T, Watabe M, Hirota S, et al. The expression of the KAI1 gene, a tumor metastasis suppressor, is directly activated by p53. Proc Natl Acad Sci USA 1998; 95:11307-11311.
18. Dong JT， Lamb PW， Rinker-Schaeffer CW，et al. KAI1,a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science 1995; 268: 884-886.
19. Dong JT, Isaaca WB, Barrett JC, et al. Genomicorganization of the human KAI1 metastasis suppressor gene.Genomics 1997, 41:25-32.
20. Metzelaar MJ,Wingaard PLJ,Peters PJ,et al.CD63 antigen.J Biol Chem 1991; 266: 3239-3245.
21. Miyake M, Koyama M, Seno M,et al.Identification of the otility-related protein (MRP-1), recognized by monoclonal antibody M31-15, which inhibits cell motility. J Exp Med 1991; 174: 1347-1354.
22. Oren R, Takahashi S, Doss C, et al .TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins.Mol Cell Biol 1990; 10:4007-4015.
23. Classon BJ, Williams AF,Willis AC,et al.The primary structure of the human leukocyte antigen CD37,a species ho.mologue of the rat MRC OX-44 antigen.J Exp Med 1998;169:1497-1502.
24. Horejsi V, Vlcek C.Novel structurally distinct family of leucocyte surface glycoproteins including CD9, CD37, CD53 and CD63.FEBS Lett 1991; 28:1-4.
25. Hotta H, Ross A.H, Huebner K, et al.Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Res 1988; 48:2955-2962.
26. Hakomori S.Aberrant Glycosylation in tumors and tumor-associated carbohydrate antigens. Adv Cancer Res 1989; 52: 257-331.
27. Ikeyama S,Koyama M, Yamaoko M, et al. Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA. J Exp Med 1993; 177: 1231-1237.
Hiroki H, Arimichi T, Takahiro T, et al.Integrin alpha3 expression as a prognostic
28. factor in colon cancer: association with MRP-1/CD9 and KAI1/CD82. Int J Cancer 2002; 97: 518-525.
29. Uchida S, Shimada Y, Watanabe G, et al. Motility-related protein (MRP-1/CD9) and KAI1/CD82 expression inversely correlate with lymph node metastasis in oesophageal squamous cell carcinoma. Br J Cancer 1999; 79: 1168-1173.
30. Sho M, Adachi M, Taki T, et al. Transmembrane 4 superfamily as a prognostic factor in pancreatic cancer. Int J Cancer 1998; 79: 509-516.
31. Huang C I, Kohno N, Ogawa E,et al.Correlation of reduction in MRP-1/CD9 and KAI1/CD82 expression with recurrences in breast cancer patients. Am J Pathol 1998; 153:973-983.
32. Adachi M, Taki T, Konishi T,et al.Novel staging protocol for non-small-cell lung cancers according to MRP-1/CD9 and KAI1/CD82 gene expression. J Clin Oncol 1998; 16:1397-1406.
33. Adachi M, Taki T, Ieki Y, et al.Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer. Cancer Res 1996; 56:1751-1755.
34. Radford K J, Mallesch J, Hersey P. Suppression of human melanoma cell growth and metastasis by the melanoma-associated antigen CD63 (ME491). Int J Cancer 1995; 62:631-635.
35. Levy S, Todd S C, Maecker H T. CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system. Annu Rev Immunol 1998; 16: 89-109.
36. Lopez GMD, Ovalle S, Yanez MM, et al.A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin. J Biol Chem 2003; 278:208-218.
37. Kirsten PL, Kristin HA, Nancy R, et al.CD53, a thymocyte selection marker whose induction requires a lower affinity TCR-MHC interaction than CD69, but is up-regulated with slower kinetics. Int Immunol 2002; 14: 249-258.
38. Engel P, Tedder T F. New CD from the B cell section of the Fifth International Workshop on Human Leukocyte Differentiation Antigens. Leuk Lymphoma 1994; 13 (Suppl 1): 61-64.
Huang CI, Kohno N, Ogawa E,et al. Correlation of reduction in MRP-1/CD9 and
39. KAI1/CD82 expression with recurrences in breast cancer patients. Am J Pathol 1998;153:973-983.
40. White A, Lamb PW, Barrett JC. Frequent downregulation of the KAI1(CD82) metastasis suppressor protein in human cancer cell lines. Oncogene 1998; 16:3143-3149.
41. Adachi M, Taki T, Konishi T,et alNovel staging protocol for non-small-cell lung cancers according to MRP-1/CD9 and KAI1/CD82 gene expression. J Clin Oncol 1998;16:1397-1406.
42. Yu Y, Yang JL, Markovic B,et al. Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers. Clin Cancer Res 1997;3:1045-1049.
43. Houle CD, Ding XY, Foley JF,et al. Loss of expression and altered localization of KAI1 and CD9 protein are associated with epithelial ovarian cancer progression. Gynecol Oncol 2002; 86:69-78.
44. Liu FS, Chen JT, Dong JT, et al. KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma. Am J Pathol 2001; 159:1629-1634
45. Liu FS, Dong JT, Chen JT, et al. KAI1 Metastasis Suppressor Protein Is Down-Regulated during the Progression of Human Endometrial Cancer.Clin Cancer Res 2003; 9:1393-1398.
46. Miyazaki T, Kato H, Shitara Y, et al. Mutation and expression of the metastasis suppressor gene KAI1 in esophageal squamous cell carcinoma. Cancer 2000;89:955-962.
47. Hinoda Y, Adachi Y, Takaoka A,et al.Decreased expression of the metastasis suppressor gene KAI1 in gastric cancer.Cancer Lett 1998;129:229-234.
48. GuoXZ, Friess H, Graber HU,et al.KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases. Cancer Res 1996;56:4876-4880.
49. Friess H, Guo XZ, Tempia-Caliera AA, et al. Differential expression of metastasis-associated genes in papilla of vater and pancreatic cancer correlates with disease stage. J Clin Oncol 2001;19:2422-2432.
50. 郭晓钟,刘民培,夏玉亭,等.KAI1基因与胰腺癌转移的关系.中华内科杂志.2000；39：626.
郭晓钟，李爱娟，刘民培，等.KAI1基因的表达与结肠癌转移的关系. 中华消化杂
51. 志.2001; 21:720-722.
52. Sun HC, Tang ZY, Zhou G,et al. KAI1 gene expression in hepatocellular carcinoma and its relationship with intrahepatic metastases. J Exp Clin Cancer Res 1998; 17:307-311.
53. Guo XZ, Friess H, Di Mola FF,et al. KAI1, a new metastasis suppressor gene, is reduced in metastatic hepatocellular carcinoma. Hepatology 1998; 28:1481-1488.
54. 陈克林,李海华,李玉梅,等.KAI1基因在7例肝癌组织中的表达.哈尔滨医科大学学报1998;32:364-365.
55. Pawlak W, Zolnierek J, Saiosiek T, et al. Antisense therapy in cancer. Cancer Treat Rev 2000; 26:333-350.
56. Raizda MK,Francis SC,Wang H,et al.Targeting of the rerin-angiotensin system by antisense gene therapy:a possible strategy for the long-term control of hypertension.J Hypertens 2000;18:353-362.
57. Roh H,Pippin JA,Green DW,et al.HER2/neu antisense targeting of human breast carcinoma.Oncogene 2000;19:6138-6143.
58. Knney JL,Guinness ME,Reiss M,et al.Antisense to the Epstein-Barrvious(EVB) -encoded latent membrane protein 1 (LMP-1) sensitizes EBV-immortalized B cells to transforming growth factor-beta and chemotherapeutic agents.Int J Cancer 2001;91:89-98.
59. Sereni D,Tubiana R,Lascousx C,et al. Pharmacokinetics and tolerability of intravenous trecovirsen(GEM91), an antisense phosphorothioate olignucleotide, in HIV-positive subjects.J Clin Pharmacol 1999;39:47-54.
60. Tian J, Tang ZY, Ye SL, et al. New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer 1999; 81:814-821.
61. Hu WL, Li YQ, He HX, et al. KAI1/CD82 gene expression in benign prostatic hyperplasia and late-stage prostate cancer in Chinese. Asian J Androl 2000; 2:221-224.
62. Guo XZ,Friess H, Shao XD,et al. KAI1 gene is differently expressed in papillary and pancreatic cancer: influence on metastasis. World J Gastroenterol 2000; 6:866-871.
63. Takaoka A, Hinoda Y, Satoh S, et al.Suppression of invasive properties of colon cancer cells by a metastasis suppressor KAI1 gene. Oncogene 1998; 16:1443-1453.
64. Liotta LA, Mandle R, Murano G, et al.Tumor cell auticrine motility factor. PNASUSA 1986, 48:3302.
65. Liotta LA.Tumor invasion and metastases: role of the basement membrane. Am J Pathol 1984; 117:339-348.
66. Albini A, Melchiori A, Santi L,et al.Tumor Tumor cell invasion inhibited by TIMP2 . J Natl cancer Inst 1991; 83:775.
67. 李楠.徐采朴.刘为纹.等.TIMP-2拮抗胃癌SGC-7901细胞侵袭能力实验研究.重庆医学. 2000;29:296-297.
68. Takahia T,Hiroshy S,Motoharm S,et al.Molecular biology of matrixmetallo -proteinase (MMPs)and tissue inhibitor of metalloproteinase (TIMPs) and the regulatons of these genes in tumor tissues.Jap J Clin Med 1995;53:179.
2. J·萨姆布鲁克,E.F.弗里奇,T·曼尼阿蒂斯著.金东雁,黎孟枫译.分子克隆实验指南.科学技术出版社出版,北京,第二版.1998.
3. 姜泊,张亚历,周殿元主编.分子生物学常用实验方法.人民军医出版社.北京.第一版.1996.
4. 张腾飞,陈诗书.γ-干拢素基因的导入及删除对人肿瘤细胞HLA-I、II类抗原表达的影响.胃肠病学1999,1:6-7.
5. F.奥斯伯,R.E.金斯顿,J.G.塞德曼等著.颜子颖,王海林译.精编分子生物学实验指南. 科学技术出版社,北京,第一版,1999.
6. 司徒镇强,吴军正主编.细胞培养.世界图书出版公司.西安.第一版.1996.
7. 朱金海,黄建富,陈燕凌等.PCNA反义寡核苷酸抑制人肝癌细胞体外增殖的研究.肝胆外科杂志.2002;10:65-68.
8. 杜月光,陈慰浙,陈智.MMP-7反义寡核苷酸对KATO Ⅲ细胞MMP-7 mRNA表达及其侵袭力的影响.中国病理生理杂志.2002,18:172-175
9. 傅建民, 余小舫, 邵永孚. 端粒酶与原发性肝癌. 世界华人消化杂志.2000; 8: 461-463.
10. 翟守恒, 刘俊彬, 刘云梅, 等.HBsAg, HCV-Ag和AFP在肝硬变和肝细胞癌组织中的表达. 世界华人消化杂志.2000;8:524-527.
11. Yang JM(杨建民),Chen WS(陈文生),Liu ZP(刘志鹏),et al.Effects of insulin-like growth factors-IR and -ⅡR antisense gene transfection on the biological behaviors of SMMC-7721 human hepatoma cells.J Gastroenterol Hepatol, 2003;18:296-301.
12. 吴孟超.中国原发性肝癌的临床和实验研究.中德临床肿瘤杂志.2002,1:1-4.
13. Zamanaka N,Okamoto E,Toyosaka A,et al.Prognostic factors after hepatectomy for hepatocellular carcinomas.Cancer 1990;65:1104-1110.
14. Shirabe K,Kanematsu T,Matsumata T,et al. Factors linked to early recurrence of small hepatocellular carcinoma after hepatectomy: univariate and multivariate analyses. Hepatology 1991; 14: 802-805.
Ikeda k,Saitou S,Tsubota A,et al.Risk factors for tumor recurrence and prognosis after
15. curative resection of hepatocellular carcinoma.Cancer 1993;71:19-25.
16. Arii S,Tanaka J,Yamazoe Y,et al.Predictive factors for intrahepatic recurrence of hepatocellular carcinoma after partial hepatectomy.Cancer 1992;69:913-919.
17. Mashimo T, Watabe M, Hirota S, et al. The expression of the KAI1 gene, a tumor metastasis suppressor, is directly activated by p53. Proc Natl Acad Sci USA 1998; 95:11307-11311.
18. Dong JT， Lamb PW， Rinker-Schaeffer CW，et al. KAI1,a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science 1995; 268: 884-886.
19. Dong JT, Isaaca WB, Barrett JC, et al. Genomicorganization of the human KAI1 metastasis suppressor gene.Genomics 1997, 41:25-32.
20. Metzelaar MJ,Wingaard PLJ,Peters PJ,et al.CD63 antigen.J Biol Chem 1991; 266: 3239-3245.
21. Miyake M, Koyama M, Seno M,et al.Identification of the otility-related protein (MRP-1), recognized by monoclonal antibody M31-15, which inhibits cell motility. J Exp Med 1991; 174: 1347-1354.
22. Oren R, Takahashi S, Doss C, et al .TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins.Mol Cell Biol 1990; 10:4007-4015.
23. Classon BJ, Williams AF,Willis AC,et al.The primary structure of the human leukocyte antigen CD37,a species ho.mologue of the rat MRC OX-44 antigen.J Exp Med 1998;169:1497-1502.
24. Horejsi V, Vlcek C.Novel structurally distinct family of leucocyte surface glycoproteins including CD9, CD37, CD53 and CD63.FEBS Lett 1991; 28:1-4.
25. Hotta H, Ross A.H, Huebner K, et al.Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Res 1988; 48:2955-2962.
26. Hakomori S.Aberrant Glycosylation in tumors and tumor-associated carbohydrate antigens. Adv Cancer Res 1989; 52: 257-331.
27. Ikeyama S,Koyama M, Yamaoko M, et al. Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA. J Exp Med 1993; 177: 1231-1237.
Hiroki H, Arimichi T, Takahiro T, et al.Integrin alpha3 expression as a prognostic
28. factor in colon cancer: association with MRP-1/CD9 and KAI1/CD82. Int J Cancer 2002; 97: 518-525.
29. Uchida S, Shimada Y, Watanabe G, et al. Motility-related protein (MRP-1/CD9) and KAI1/CD82 expression inversely correlate with lymph node metastasis in oesophageal squamous cell carcinoma. Br J Cancer 1999; 79: 1168-1173.
30. Sho M, Adachi M, Taki T, et al. Transmembrane 4 superfamily as a prognostic factor in pancreatic cancer. Int J Cancer 1998; 79: 509-516.
31. Huang C I, Kohno N, Ogawa E,et al.Correlation of reduction in MRP-1/CD9 and KAI1/CD82 expression with recurrences in breast cancer patients. Am J Pathol 1998; 153:973-983.
32. Adachi M, Taki T, Konishi T,et al.Novel staging protocol for non-small-cell lung cancers according to MRP-1/CD9 and KAI1/CD82 gene expression. J Clin Oncol 1998; 16:1397-1406.
33. Adachi M, Taki T, Ieki Y, et al.Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer. Cancer Res 1996; 56:1751-1755.
34. Radford K J, Mallesch J, Hersey P. Suppression of human melanoma cell growth and metastasis by the melanoma-associated antigen CD63 (ME491). Int J Cancer 1995; 62:631-635.
35. Levy S, Todd S C, Maecker H T. CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system. Annu Rev Immunol 1998; 16: 89-109.
36. Lopez GMD, Ovalle S, Yanez MM, et al.A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin. J Biol Chem 2003; 278:208-218.
37. Kirsten PL, Kristin HA, Nancy R, et al.CD53, a thymocyte selection marker whose induction requires a lower affinity TCR-MHC interaction than CD69, but is up-regulated with slower kinetics. Int Immunol 2002; 14: 249-258.
38. Engel P, Tedder T F. New CD from the B cell section of the Fifth International Workshop on Human Leukocyte Differentiation Antigens. Leuk Lymphoma 1994; 13 (Suppl 1): 61-64.
Huang CI, Kohno N, Ogawa E,et al. Correlation of reduction in MRP-1/CD9 and
39. KAI1/CD82 expression with recurrences in breast cancer patients. Am J Pathol 1998;153:973-983.
40. White A, Lamb PW, Barrett JC. Frequent downregulation of the KAI1(CD82) metastasis suppressor protein in human cancer cell lines. Oncogene 1998; 16:3143-3149.
41. Adachi M, Taki T, Konishi T,et alNovel staging protocol for non-small-cell lung cancers according to MRP-1/CD9 and KAI1/CD82 gene expression. J Clin Oncol 1998;16:1397-1406.
42. Yu Y, Yang JL, Markovic B,et al. Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers. Clin Cancer Res 1997;3:1045-1049.
43. Houle CD, Ding XY, Foley JF,et al. Loss of expression and altered localization of KAI1 and CD9 protein are associated with epithelial ovarian cancer progression. Gynecol Oncol 2002; 86:69-78.
44. Liu FS, Chen JT, Dong JT, et al. KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma. Am J Pathol 2001; 159:1629-1634
45. Liu FS, Dong JT, Chen JT, et al. KAI1 Metastasis Suppressor Protein Is Down-Regulated during the Progression of Human Endometrial Cancer.Clin Cancer Res 2003; 9:1393-1398.
46. Miyazaki T, Kato H, Shitara Y, et al. Mutation and expression of the metastasis suppressor gene KAI1 in esophageal squamous cell carcinoma. Cancer 2000;89:955-962.
47. Hinoda Y, Adachi Y, Takaoka A,et al.Decreased expression of the metastasis suppressor gene KAI1 in gastric cancer.Cancer Lett 1998;129:229-234.
48. GuoXZ, Friess H, Graber HU,et al.KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases. Cancer Res 1996;56:4876-4880.
49. Friess H, Guo XZ, Tempia-Caliera AA, et al. Differential expression of metastasis-associated genes in papilla of vater and pancreatic cancer correlates with disease stage. J Clin Oncol 2001;19:2422-2432.
50. 郭晓钟,刘民培,夏玉亭,等.KAI1基因与胰腺癌转移的关系.中华内科杂志.2000；39：626.
郭晓钟，李爱娟，刘民培，等.KAI1基因的表达与结肠癌转移的关系. 中华消化杂
51. 志.2001; 21:720-722.
52. Sun HC, Tang ZY, Zhou G,et al. KAI1 gene expression in hepatocellular carcinoma and its relationship with intrahepatic metastases. J Exp Clin Cancer Res 1998; 17:307-311.
53. Guo XZ, Friess H, Di Mola FF,et al. KAI1, a new metastasis suppressor gene, is reduced in metastatic hepatocellular carcinoma. Hepatology 1998; 28:1481-1488.
54. 陈克林,李海华,李玉梅,等.KAI1基因在7例肝癌组织中的表达.哈尔滨医科大学学报1998;32:364-365.
55. Pawlak W, Zolnierek J, Saiosiek T, et al. Antisense therapy in cancer. Cancer Treat Rev 2000; 26:333-350.
56. Raizda MK,Francis SC,Wang H,et al.Targeting of the rerin-angiotensin system by antisense gene therapy:a possible strategy for the long-term control of hypertension.J Hypertens 2000;18:353-362.
57. Roh H,Pippin JA,Green DW,et al.HER2/neu antisense targeting of human breast carcinoma.Oncogene 2000;19:6138-6143.
58. Knney JL,Guinness ME,Reiss M,et al.Antisense to the Epstein-Barrvious(EVB) -encoded latent membrane protein 1 (LMP-1) sensitizes EBV-immortalized B cells to transforming growth factor-beta and chemotherapeutic agents.Int J Cancer 2001;91:89-98.
59. Sereni D,Tubiana R,Lascousx C,et al. Pharmacokinetics and tolerability of intravenous trecovirsen(GEM91), an antisense phosphorothioate olignucleotide, in HIV-positive subjects.J Clin Pharmacol 1999;39:47-54.
60. Tian J, Tang ZY, Ye SL, et al. New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer 1999; 81:814-821.
61. Hu WL, Li YQ, He HX, et al. KAI1/CD82 gene expression in benign prostatic hyperplasia and late-stage prostate cancer in Chinese. Asian J Androl 2000; 2:221-224.
62. Guo XZ,Friess H, Shao XD,et al. KAI1 gene is differently expressed in papillary and pancreatic cancer: influence on metastasis. World J Gastroenterol 2000; 6:866-871.
63. Takaoka A, Hinoda Y, Satoh S, et al.Suppression of invasive properties of colon cancer cells by a metastasis suppressor KAI1 gene. Oncogene 1998; 16:1443-1453.
64. Liotta LA, Mandle R, Murano G, et al.Tumor cell auticrine motility factor. PNASUSA 1986, 48:3302.
65. Liotta LA.Tumor invasion and metastases: role of the basement membrane. Am J Pathol 1984; 117:339-348.
66. Albini A, Melchiori A, Santi L,et al.Tumor Tumor cell invasion inhibited by TIMP2 . J Natl cancer Inst 1991; 83:775.
67. 李楠.徐采朴.刘为纹.等.TIMP-2拮抗胃癌SGC-7901细胞侵袭能力实验研究.重庆医学. 2000;29:296-297.
68. Takahia T,Hiroshy S,Motoharm S,et al.Molecular biology of matrixmetallo -proteinase (MMPs)and tissue inhibitor of metalloproteinase (TIMPs) and the regulatons of these genes in tumor tissues.Jap J Clin Med 1995;53:179.