FHIT基因转染对肝癌细胞系HepG2作用.docVIP

FHIT基因转染对肝癌细胞系HepG2作用 　【关键词】 肝癌 Role of transfection of FHIT gene in human hepatocellular carcinoma cell line HepG2 　　【Abstract】 AIM: To study the role of transfection of FHIT gene into a human liver cancer cell line HepG2. METHODS: FHIT gene was transfected into human liver cancer cell line HepG2 by lipofectamine. After selection with G418, resistant colonies were obtained. The overexpression of FHIT was detected by immunochemistry and Western blotting methods. Apoptosis and cell cycle were analyzed by flow cytometry (FCM). The morphological changes were observed by light and electron microscope. RESULTS: Stable expression of FHIT protein in transfected HepG2 cells was obtained and the growth rate of these transfected HepG2 was inhibited. Early apoptosis was observed under transmission electron microscope and flow cytometry showed that the progression of cell cycle was arrested from S phase to G2 phase. CONCLUSION: The results indicate that FHIT gene can inhibit the growth and proliferation of HepG2 by inducing its apoptosis and cell cycle arrest in vitro. 　　【Keywords】 FHIT； gene transfer； apoptosis; hepatocarcinoma 　　【摘要】 目的：探讨外源性FHIT基因的表达对肝癌细胞株HepG2的影响. 方法：用脂质体介导法将pcDNA3.1FHIT及空载体pcDNA3.1(+)导入肝癌细胞系HepG2中，G418选择培养，经免疫组化和Western blotting鉴定其过表达. 用流式细胞仪、普通光镜、透射电镜观察FHIT基因的表达对HepG2细胞凋亡和增殖的影响. 结果：获得FHIT基因稳定表达的肝癌细胞株HepG2FHIT. 转染FHIT基因的HepG2细胞的生长速率明显下降,电镜下超微结构发生了凋亡早期改变，流式分析细胞的生长周期可见S／G2期阻滞. 结论：外源性FHIT基因在体外通过诱导其凋亡及细胞周期俘获作用可有效抑制HepG2生长增殖. 　　【关键词】 FHIT基因; 基因转染；凋亡；肝癌 　　0引言 　　脆性组氨酸三联体（fragile histidine triad, FHIT）基因是一个新的抑癌基因，在多种肿瘤中存在转录异常和表达缺失. FHIT基因对细胞生长的调控和凋亡的诱导不同于经典的抑癌基因，FHIT基因只出现纯合子或半合子的缺失，只有少数肿瘤中可检测到点突变，但其具体的抑癌机制还不是很清楚. 我们将FHIT基因的真核表达载体pcDNA3.1FHIT转染至人肝癌细胞系HepG2中，并得到稳定的过表达，从细胞形态学和细胞周期方面探讨FHIT基因对肝癌HepG2细胞的影响. 　　1材料和方法 　　1.1材料 　　质粒pcDNA3.1FHIT由本室构建，大肠杆菌DH5α由第四军医大学生化教研室李云峰硕士惠赠，HepG2由本校病理教研室严小昱硕士惠赠；小量质粒提取试剂盒购自Omega公司； FHIT兔抗人多克隆抗体、二抗购自北京中山公司；Lipofectamine 2000购自Invitrogen公司，其他生化试剂分析纯. 　　1.2方法 　　参照杨安钢等［1］所述，制备感受态大肠杆菌DH5α，质粒pcDNA3.1FHIT转化细菌后进行扩菌,按Omega公司小量质粒提取试剂盒说明提取、纯化质粒DNA，