乙肝病毒X蛋白对肝前体细胞分化的影响

Effect of hepatitis B virus X protein mediated by adenovirus on the differentiation of hepatic progenitor cells

目的:检测乙肝病毒X(Hepatitis B virus X,HBx)蛋白对肝前体细胞分化的影响,以探讨HBx蛋白能否促进肝干细胞的恶性转化。方法:以腺病毒Ad-HBx和Ad-GFP感染肝前体细胞株Hp14-19,RT-PCR和Western blot证实HBx蛋白在肝前体细胞内的表达。RT-qPCR和免疫荧光检测早期和晚期分化指标的变化,并于诱导后11 d用PAS染色观察细胞的分化成熟程度。结果:Ad-HBx能有效感染肝前体细胞株Hp14-19并表达。与对照组(感染Ad-GFP)相比,实验组(感染Ad-HBx)细胞分化早期指标穿膜蛋白(Delta like homolog,DLK)、肌酸激酶19(Creatine kinase19,CK19)和甲胎蛋白(Alpha fetal protein,AFP)表达降低减慢,而晚期指标CK18和白蛋白(Albumin,ALB)表达升高不明显。PAS染色阳性率降低。差异具有统计学意义(P<0.05)。结论:HBx可抑制肝前体细胞的分化,这可能是最终导致肝前体细胞恶性转化的原因之一。

Objective：To detect the effect of hepatitis B virus X（HBx） protein on the differentiation of hepatic progenitor cell and to explore whether HBx protein can promote the malignant transformation of hepatic stem cell. Methods ： The hepatic progenitor cell Hp14-19 was infected with adenovirus Ad-HBx and Ad-GFP. The expressions of HBx protein in hepatic progenitor cell were confirmed by RT-PCR and Western blot. The changes of differentiation marker alpha fetal protein（AFP）, delta like homolog（DLK）, creatine kinase19（CK19）, CK18 and albumin（ALB） in the hepatic progenitor cell treated with HBx were evaluated by RT-qPCR and immunofluorescence. The morphological change was observed by PAS staining at 11 d after the inducement. Results：HBx was expressed in the hepatic progenitor cell Hp14-19 infected with Ad-HBx. Compared with those in control group（infected with Ad-GFP）, the expressions of early marker DLK, CK19 and AFP were decreased slowly while later marker CK18, ALB were increased slowly in experiment group（infected with Ad-HBx）. PAS staining results showed that HBx inhibited glycogen synthesis in HP14.5 cells. Conclusions：HBx can inhibit the differentiation of hepatic progenitor cell, which maybe involved in the tumorigenesis of HBx-mediated liver cancer.