【作者】 司遂海；

【导师】 杨建民；

【作者基本信息】 第三军医大学 ， 内科学， 2003， 硕士

【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。更多还原

【Abstract】 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<WP=6>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.更多还原