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目的 探讨丙肝病毒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 (PBK HCVc) ,经酶切鉴定后 ,将其转染到胆管癌细胞(QBC939)中 ,经G4 18选择培养 ,免疫细胞学、Westernblotting鉴定其表达 ;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 PCR、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QBC939细胞中有稳定表达。透射电镜见胞质空泡内球型病毒颗粒 ,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 本实验为进一步探讨丙肝病毒感染在胆管癌中的致癌机理提供了理想的细胞株模型 ,NF κB可能与丙肝病毒感染的胆管癌细胞免疫逃逸及致癌有关。
Objective To investigate the carcinogenic mechanism of hepatitis C virus infection in cholangiocarcinoma. Methods The HCV C gene recombinant plasmid (PBK HCVc) was constructed by molecular cloning technique. The recombinant plasmid was identified by restriction enzyme digestion and transfected into cholangiocarcinoma cells (QBC939). The recombinant plasmid was selected by G418, immunocytochemistry and Western blotting were used to identify the expression. The morphological changes of cells were observed by transmission electron microscope. The expression of NF-κB was detected by immunocytochemistry. Results PCR and restriction endonuclease analysis showed that the HCV C gene plasmid was successfully constructed. The constructed PBK HCVc plasmid was stably expressed in QBC939 cells by immunocytochemistry and Western blotting. Transmission electron microscopy was seen cytoplasmic vacuolar vesicular virus particles, the virus particles about 50 - 80nm, with the outer membrane structure. HCV core protein activates NFKB expression. Conclusion This experiment provides an ideal cell model for further exploring the carcinogenic mechanism of hepatitis C virus infection in cholangiocarcinoma. NF κB may be related to immune escape and carcinogenesis of hepatitis C virus-infected cholangiocarcinoma cells.