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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 study the effects of hepatitis B virus (HBV) X protein (HBx) on the apoptosis of mouse embryonic liver stem cells (ELSC) and the expression of Bcl2, Mcl1 and Bax. Methods HBx gene was transfected into mouse embryonic stem cell line ELSC14.5 using adenovirus vector expressing green fluorescent protein (GFP). RT-PCR and Western blot were used to detect the mRNA transcription of HBx gene The expression of Bcl-2, Mcl1 and Bcl-2 mRNA was detected by Hoechst33342 staining, TUNEL and flow cytometry were used to detect the cell apoptosis. Real-time PCR and Western blot were used to detect the expression of Bax, Transcription level and protein expression level. Results The recombinant adenovirus Ad-GFP-HBx could effectively infect ELSC14.5 cells, and the expression of HBx gene and protein could be expressed specifically. The infected cells showed shrinkage and the number of marginalized cells was decreased and the apoptosis rate was decreased The mRNA and protein expression levels of Bcl2 and Mcl1 in the cells increased, while the expression of Bax decreased. Conclusion HBx can inhibit the apoptosis and promote the survival of mouse embryonic hepatic stem cells by regulating the imbalance of anti-apoptotic factor and pro-apoptotic factor in Bcl2 family.