HCV核心基因转染胆管癌细胞中NF-κB的表达

NF-κB expression in cholangiocarcinoma transfected with hepatitis C virus core gene

目的 探讨丙型肝炎病毒 (HCV)感染在胆管癌中的致癌机理。方法 通过分子克隆技术构建HCV C基因重组质粒 ,经酶切鉴定后 ,转染胆管癌细胞 (QBC93 9) ,经G418选择培养 ,以免疫细胞学测定、Westernblotting鉴定其表达 ;以透射电镜观察细胞形态学改变 ,并用免疫细胞学检测NF κB的表达。结果 限制性内切酶反应证明HCV C基因质粒构建成功 ,构建的PBK HCVc质粒经免疫细胞学、Westernblotting鉴定在QBC93 9细胞中有稳定表达。透射电镜可见胞质空泡内球型病毒颗粒 ,直径约 5 0～ 80nm ,有外膜结构。HCV核心蛋白可激活NF κB表达。结论 为进一步探讨HCV感染在胆管癌中的致癌机理提供了理想的实验细胞株 ,NF κB可能与HCV感染的胆管癌细胞免疫逃逸及致癌有关。

Objective To study the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma. Methods Recombinant plasmid of HCV-C gene constructed by molecular cloning technique was identified with restricting enzyme map. Then, it was transfected into QBC 939 cells with lipofectin. After selection with G418, the resistant colonies were obtained and analysed by immunocytochemistry and Western blotting. Their morphology was observed by transmission electron microscopy (TEM). The expression of NF-κB was detected by immunocytochemistry.Results The results suggested that the recombinant plasmid was proved to carry the target gene by restricting enzyme map. Moreover, it could express HCV-C protein efficiently in QBC 939 cells. The HCV-like particles were found in the cytoplasm by TEM, which were spherical with diameter of 50～80 nm possessing an outer membrane. Moreover, NF-κB activation was shown in HCV core gene-transfected cells.Conclusion Because HCV-C gene could express steadily in cholangiocarcinoma cells, the transfected tumor cells (QBC 939 -HCV C) are an experimental model for studying the effect of HCV on the development of cholangiocarcinoma. The activation of NF-κB may be related to escape from immune surveillance and carcinogenesis of cholangiocarcinoma.