论文部分内容阅读
目的建立乙型肝炎病毒X基因-丙型肝炎病毒C基因(HBV X-HCV C)共表达HepG2细胞模型,并探讨其对血管内皮细胞生长因子表达的影响。方法双酶切质粒pXT1-X,得到完整的HBV X基因片段后,将其插入到质粒PBK CMV和PBK-HCV C的相应酶切位点,得到重组质粒PBK-X和PBK-X-C;再将质粒PBK-CMV、PBK-X、 PBK-HCV C和PBK-X-C分别导入肝癌细胞株HepG2中,G418筛选,逆转录聚合酶链反应、Western blot鉴定HBV X和HCV C蛋白表达;免疫组织化学、Western blot检测血管内皮细胞生长因子蛋白质表达。结果质粒PBK-CMV、 PBK-X、PBK-HCV C和PBK X-C在HepG2细胞中有稳定表达。共表达HBV X-HCV C蛋白的细胞血管内皮细胞生长因子蛋白质表达较转染空载体的细胞及单独表达HBV X、HCV C蛋白的细胞明显升高。结论HBV X-HCV C共表达能显著上调血管内皮细胞生长因子蛋白质表达,提示HBV、HCV可能具有协同致癌作用。
Objective To establish a HepG2 cell model co-expressing hepatitis B virus X gene and hepatitis C virus C gene (HBV X-HCV C), and to explore its effect on the expression of vascular endothelial growth factor. Methods Double enzyme digestion of plasmid pXT1-X resulted in the complete HBV X gene fragment inserted into plasmids PBK CMV and PBK-HCV C corresponding restriction sites to obtain recombinant plasmids PBK-X and PBK-XC; The plasmids PBK-CMV, PBK-X, PBK-HCV C and PBK-XC were respectively introduced into HepG2 cells. The expression of HBV X and HCV C were identified by G418 screening, reverse transcriptase polymerase chain reaction and Western blot. Immunohistochemistry, Western blot was used to detect the protein expression of vascular endothelial growth factor. Results Plasmids PBK-CMV, PBK-X, PBK-HCV C and PBK X-C were stably expressed in HepG2 cells. Compared with the cells transfected with empty vector and the cells expressing HBV X and HCV C alone, the expression of the protein of vascular endothelial growth factor co-expressing HBV X-HCV C protein was significantly increased. Conclusion The co-expression of HBV X-HCV C can up-regulate the expression of VEGF protein, suggesting that HBV and HCV may have a synergistic carcinogenic effect.