乙型肝炎病毒X蛋白对小鼠胚胎肝干细胞凋亡及相关蛋白表达的影响

Effect of hepatitis B virus X protein on apoptosis of mouse embryonic liver stem cells and expression of associated proteins

目的研究乙型肝炎病毒(hepatitis B virus,HBV)X蛋白(HBx)对小鼠胚胎肝干细胞(embryonic liver stemcell,ELSC)凋亡及相关蛋白Bcl2、Mcl1、Bax表达的影响。方法采用表达绿色荧光蛋白(green fluorescent protein,GFP)的腺病毒载体系统将HBx基因转入小鼠胚胎肝干细胞ELSC14.5中,采用RT-PCR和Western blot法检测细胞中HBx基因mRNA的转录和蛋白的表达;Hoechst33342染色法观察细胞核的改变;TUNEL法和流式细胞术检测细胞的凋亡情况;Real-time PCR和Western blot法检测抗凋亡因子Bcl2、Mcl1和促凋亡因子Bax基因mRNA的转录水平和蛋白的表达水平。结果重组腺病毒Ad-GFP-HBx能有效感染ELSC14.5细胞,HBx基因和蛋白均能特异性表达;感染的ELSC14.5细胞核呈现固缩,且边缘化的细胞数减少,细胞凋亡率降低;细胞中Bcl2和Mcl1基因mRNA的转录水平和蛋白的表达水平均增高,而Bax的表达降低。结论 HBx可通过调节Bcl2家族中抗凋亡因子和促凋亡因子的比例失衡,来抑制小鼠胚胎肝干细胞的凋亡,促进其存活。

Objective To investigate the effect of hepatitis B virus X protein（HBx）on apoptosis of mouse embryonic liver stem cells（ELSCs）and expression of associated proteins.Methods ELSC14.5 cells were infected with recombinant adenovirus Ad-GFP-HBx,and determined for mRNA transcription and protein expression of HBx by RT-PCR and Western blot respectively,for change of nuclei by Hoechst33342 staining,and for apoptosis by TUNEL method and flow cytometry.The mRNA transcription levels of anti-apoptotic factors Bcl2 and Mcl1 as well as apoptosis-promoting factor Bax were determined by Real-time PCR,while the protein expression levels by Western blot.Results ELSC14.5 cells were effectively infected with Ad-GFP-HBx,and HBx was expressed specifically at both mRNA and protein levels.Nuclear condensation and chromatin marginization were observed in infected ELSC14.5 cells,while the apoptotic rate of cells decreased.The mRNA transcription and protein expression levels of Mcl1 and Bcl2 in infected cells increased,while those of Bax decreased.Conclusion HBx inhibited the apoptosis and encouraged the survival of mouse ELSCs by regulating the unbalanced proportion of anti-apoptotic and apoptosis-promoting factors in Bcl2 family.