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目的探讨乙型肝炎病毒X蛋白(HBx)诱导大鼠肾小管上皮细胞凋亡及其可能的机制。方法以HBx的表达载体转染体外培养的大鼠肾小管上皮细胞(NRK-52E)建立HBx过表达模型。分别转染空质粒pc-DNA3.1(+)24h(空质粒转染组),以及转染pc-DNA3.1(+)HBx质粒24h(HBx24h组)、48h(HBx48h组)和72h(HBx72h组),并设对照组(未做转染)。应用流式细胞仪测定细胞凋亡率。分别采用Western印迹法检测Fas、Fas配体(FasL)、Bcl-2、Bax的蛋白质表达水平,分光光度测定法测定半胱氨酸天冬氨酸蛋白酶(Caspase)8活性。结果 HBx24h组、HBx48h组、HBx72h组的HBx、Fas、FasL、Bax的蛋白质相对表达量,以及Caspase8的相对活性逐渐增加,且组间差异均有统计学意义(P值分别<0.05、0.01);Bcl-2的蛋白质相对表达量逐渐降低,组间差异亦有统计学意义(P值分别<0.05、0.01)。对照组与空质粒转染组间细胞凋亡率的差异无统计学意义(P>0.05);HBx24h组、HBx48h组和HBx72h组的细胞凋亡率逐渐升高,组间差异有统计学意义(P值均<0.05),且均显著高于对照组和空质粒转染组(P值均<0.05)。结论 HBx基因通过Fas/FasL死亡受体途径促进NRK-52E细胞凋亡,Caspase8的活化可能参与其中。
Objective To investigate the apoptosis of rat renal tubular epithelial cells induced by hepatitis B virus X protein (HBx) and its possible mechanism. Methods HBx expression vector was transfected into cultured rat renal tubular epithelial cells (NRK-52E) to establish HBx overexpression model. (HBx24h group), 48h (HBx48h group) and 72h (HBx72h group) were transfected with pc-DNA3.1 (+ Group), and control group (not transfection). Flow cytometry was used to determine apoptosis rate. Western blotting was used to detect the protein expression of Fas, Fas ligand, Bcl-2 and Bax, respectively. The activity of caspase 8 was determined by spectrophotometry. Results The protein expression of HBx, Fas, FasL and Bax in HBx24h group, HBx48h group and HBx72h group increased gradually, and the relative activity of Caspase8 increased gradually with statistical significance (P <0.05, 0.01). The relative expression of Bcl-2 protein decreased gradually, with significant difference between groups (P <0.05, 0.01 respectively). There was no significant difference in apoptosis rate between control group and empty plasmid transfection group (P> 0.05). The apoptosis rate of HBx24h group, HBx48h group and HBx72h group increased gradually, the difference was statistically significant (P < P <0.05), which were significantly higher than those in control group and empty plasmid transfected group (all P <0.05). Conclusion The HBx gene can promote the apoptosis of NRK-52E cells through the Fas / FasL death receptor pathway, and the activation of Caspase8 may be involved.