乙型肝炎病毒/P22蛋白对HepG2细胞凋亡的影响

目的研究HBV／P22蛋白对肿瘤坏死因子（TNF）α诱导的HepG2细胞凋亡的影响。方法用放线菌素D和TNFo【分别诱导表达HBV／P22蛋白的HepG2细胞（实验组）、表达空载体的HepG2细胞（阴性对照组）和HepG2细胞（空白对照组）凋亡，雅培试剂检测细胞上清液中HBeAg的表达，Westernblot及免疫细胞化学法检测HBV／P22蛋白表达，流式细胞仪和末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记（TUNEL）法检测细胞凋亡。体内实验：将前述三种细胞分别注射入裸鼠皮下，放线菌素D、TNF仅注射瘤体后，免疫组织化学法检测组织HBV／P22蛋白的表达，TUNEL法检测组织细胞凋亡率。结果实验组细胞培养上清液中HBeAg表达阳性，两对照组阴性；Westernblot及免疫细胞化学法检测均显示实验组细胞有HBV／P22蛋白表达，两对照组均为阴性；流式细胞仪和TUNEL结果均显示实验组细胞凋亡率明显低于两对照组（P〈0．05）。体内实验：实验组瘤体组织免疫组织化学检测结果显示HBV／P22蛋白表达阳性，TUNEL检测结果显示接种表达HBV／P22蛋白的HepG2细胞裸鼠瘤组织细胞凋亡率明显低于两对照组（P〈0．05）。结论HBV／P22蛋白在体内外均可抑制TNFα诱导的HepG2细胞凋亡。

The influence of HBV/P22 protein on the apoptosis of HepG2 cells:an experimental study

OBJECTIVE: To study the influence of HBV/P22 protein on the induced apoptosis of HepG2 cells. METHODS: In vitro, two HepG2 strains were transfected with pcDNA3.1+ and pcDNA3.1+HBV/P22 respectively and the cells were exposed to Act D and TNF alpha for 6h and then the induced apoptosis was detected by flow cytometry (FCM) and TUNEL technique. Supernatant HBeAg was detected by Abbott reagent. The intracellular expression of HBV/P22 protein was measured by Western blot and immunochemistry. In vivo, three cell groups were inoculated into nude mice by subcutaneous injections. After two weeks, Act D and TNF alpha were injected into the tumors and then the induced apoptosis in the tissues was detected by TUNEL technique. The expression of HBV/P22 protein in the tumor tissues was detected by immunochemistry. RESULTS: In vitro, in HepG2- pcDNA3.1+HBV/P22 cells, supernatant HBeAg was positive and intracellular HBV/P22 protein was positively expressed. The apoptosis proportion of HepG2-pCDNA3.1+HBV/P22 cells was markedly lower than HepG2 and HepG2-pcDNA3.1+ cells (P < 0.05). In vivo, HBV/P22 protein was expressed in the tumor tissues, and the apoptosis proportion in the group injected with HepG2-pcDNA3.1+HBV/P22 cells was markedly lower than those injected with HepG2 and HepG2-pcDNA3.1+cells (P < 0.05). CONCLUSION: HBV/P22 protein could inhibit the induced apoptosis of HepG2 cells both in vitro and in vivo.