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目的 探讨丙型肝炎病毒核心蛋白 (HCV -C)对人胆管上皮细胞 (BEC)的转化及致癌作用。方法 通过脂质体介导将含有HCV- C基因重组真核表达载体pcDNAHCV- C导入BEC细胞,G418筛选表达目的基因的细胞;聚合酶链反应 (PCR)及免疫组织化学SABC法检测细胞中HCV -C基因及蛋白的表达;细胞计数、锚着非依赖性生长实验、成瘤性检测等鉴定其生物学行为变化,免疫组织化学SABC法检测所致肿瘤组织中HCV- C蛋白表达。结果 HCV -C转染的BEC细胞中HCV- C蛋白表达于胞质,质粒pcDNAHCV C转染细胞的倍增时间较pcDNA3转染细胞和未转染BEC细胞明显缩短(分别为 14h, 28h, 30h)。pcDNAHCV C和pcDNA3转染细胞及未转染BEC在软琼脂中的克隆形成率分别为 36. 0%、2 .5%和 1 5% (P<0. 01)。3种细胞接种裸鼠后,pcDNAHCV C转染细胞注射组出现肿瘤,病理学检查证实为胆管细胞癌,肿瘤组织有HCV- C蛋白的表达。而pcDNA3转染细胞及未转染BEC细胞注射组在注射 36d后仍未见肿瘤发生。结论 HCV- C蛋白具有促胆管细胞转化和促进肿瘤发生的作用。
Objective To investigate the transformation and carcinogenesis of hepatitis C virus core protein (HCV-C) on human cholangiocarcinoma (BEC). Methods The recombinant eukaryotic expression vector pcDNAHCV-C containing HCV-C gene was introduced into BEC cells by lipofectamine. The cells expressing the target gene were screened by G418. The expression of HCV in the cells was detected by polymerase chain reaction (PCR) and immunohistochemical SABC C gene and protein expression; cell count, anchorage-independent growth assay, tumorigenicity test to identify changes in their biological behavior, immunohistochemical SABC method to detect the resulting tumor tissue HCV-C protein expression. Results HCV-C protein expression in the cytoplasm of HCV-C transfected BEC cells was significantly shorter than that in pcDNA3-transfected and non-transfected BEC cells (14h, 28h and 30h respectively) . The clonogenic rates of pcDNAHCV C and pcDNA3 transfected cells and non-transfected BEC in soft agar were 36.0%, 2.5% and 15%, respectively (P <0.01). After the three kinds of cells were inoculated with nude mice, tumor was found in the injected group of pcDNAHCV C transfected cells, and the expression of HCV-C protein was confirmed in cholangiocarcinoma and tumor tissues by pathological examination. The pcDNA3 transfected cells and non-transfected BEC cells injection group after injection 36d still no tumor. Conclusion HCV-C protein has the function of transforming cholangiocarcinoma cells and promoting tumorigenesis.