HBx基因抑制肝前体细胞凋亡与分化的小鼠体内研究

目的 构建将乙肝病毒X(hepatitis B virus X,HBx)基因导入小鼠肝脏内的动物模型,探讨HBx蛋白对小鼠体内肝前体细胞凋亡与分化的影响.方法 从小鼠肝门静脉注射携带HBx基因的肝前体细胞和生理盐水,于第30、60、90、120天收取小鼠肝前体细胞标本,通过RT-PCR和Western blot 验证HBx基因在肝前体细胞中的表达;通过荧光显微镜、免疫组织化学染色法、RT-PCR检测细胞株是否到达小鼠肝脏并表达目的基因;使用TUNEL-FITC/DAPI法、RT-PCR和Western blot检测肝前体细胞的凋亡和分化情况.结果 携带了HBx基因的肝前体细胞株成功到达小鼠肝脏内并表达HBx蛋白,TUNEL-FITC/DAPI法显示HBx显著抑制小鼠肝前体细胞发生凋亡,RT-PCR和Western blot结果显示,抗凋亡蛋白Bcl2、Mcl-1的表达上调,促凋亡蛋白Bax的表达下调,细胞分化早期指标CK19表达增加,而细胞晚期分化指标CK18表达降低,差异均具有统计学意义(P＜0.05).结论 HBx蛋白在小鼠体内可以抑制肝前体细胞凋亡和分化成熟,诱发肝癌发生的早期事件.

HBx gene inhibits apoptosis and differentiation of hepatic progenitor cells in mice

Objective To establish a mouse model of transfecting hepatitis B virus X (HBx) into the liver, and determine the effect of HBx on the apoptosis and differentiation of hepatic cells in vivo.Methods Hepatic progenitor cells (HPCs) carrying HBx gene or normal saline were respectively injected into the portal vein of mice.On days 30, 60, 90 and 120 after injection, all mice were sacrificed and the liver tissues were collected.The expression of HBx in the HPCs was detected by RT-PCR and Western blotting.Fluorescence microscopy, immunohistochemical staining and RT-PCR were used to detect whether HPCs reached the liver and expressed the target gene.TUNEL-FITC/DAPI assay, RT-PCR and Western blotting were employed to detect the apoptosis and differentiation of HPCs.Results HPCs carrying HBx gene reached the liver of mice and expressed HBx successfully.TUNEL-FITC/DAPI assay showed that HBx significantly inhibited the apoptosis of the HPCs in the mice.RT-PCR and Western blotting showed that the expression levels of antiapoptotic proteins Bcl2 and Mcl-1 were up-regulated and the expression of pro-apoptotic protein Bax was downregulated in the liver tissues.The expression of early differentiation marker CK19 were increased while later marker CK18 were decreased, with significant differences (P ＜ 0.05).Conclusion HBx protein inhibits the apoptosis and differentiation of HPCs in mice and induces early events of the occurrence of liver cancer.