泛素特异性蛋白酶7对乙肝细胞中凋亡因子的调控作用研究

Study on the regulatory effect of ubiquitin specific protease 7 on apoptotic factors in hepatitis B cells

目的研究泛素特异性蛋白酶7(USP7)在肝细胞中对凋亡因子的调控作用。方法将HepG2.2.15细胞随机分为3组:对照组,转染组和阴性对照组。对照组为常规培养的细胞;转染组转染si-USP7,阴性对照组使用10%siRNA-mate转染试剂进行处理,各组均处理72 h。实时荧光定量聚合酶链反应(Q-PCR)检测USP7、P53、B淋巴细胞瘤-2相关X蛋白(Bax)的mRNA表达水平;蛋白质印迹法检测USP7、P53蛋白的表达情况;酶联免疫法检测谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乙型肝炎病毒(HBV)DNA、乙型肝炎表面抗原(HBsAg)和乙型肝炎核心e抗原(HBeAg)的表达量。结果对照组、转染组、阴性对照组USP7 mRNA表达水平分别为1.74±0.01,1.00±0.06和1.73±0.01;P53 mRNA表达水平分别为1.94±0.01,0.81±0.03和1.92±0.01;Bax mRNA表达水平分别为6.28±0.04,1.81±0.16,6.26±0.04;蛋白结果的趋势与基因一致。以上3组的HBV-DNA表达量分别为(1032.68±2.96),(541.92±6.78)和(1035.44±1.86)pg·mL^(-1);HBsAg表达量分别为(905.19±1.41),(246.23±7.04)和(902.56±4.74)U·mL^(-1);HBeAg表达量分别为(45.28±0.06),(10.09±0.68)和(45.82±0.36)U·mL^(-1)。以上指标,转染组与对照组比,差异均有统计学意义(均P<0.05)。结论USP7的下调能减少P53的表达,使Bax表达下降,并降低乙型肝炎病毒的复制和细胞凋亡。

Objective To study the regulation of ubiquitin specific protease 7(USP7)on apoptosis factors in hepatocytes.Methods HepG2.2.15 cells were randomly divided into control group,transfection group and negative control group.The control group was conventionally cultured cells;the transfection group was transferred with si-USP7,and the negative control group was treated with 10%siRNA-mate transfection reagent,and each group was treated for 72 h.Real-time fluorescent quantitative polymerase chain reaction was used to detect the mRNA expression levels of USP7,P53,B lymphocyte tumor-2 associated X protein(Bax).Western blotting was used to detect the expression of USP7 and P53 proteins;the expression of glutamic pyruvic transaminase(GPT),glutamic-oxalacetic transaminase(GOT),hepatitis B virus(HBV)DNA,hepatitis B surface antigen(HBsAg)and hepatitis Be antigen(HBeAg)were detected by enzyme linked immunosorbent assay.Results The expression levels of USP7 mRNA in control group,transfection group and negative control group were 1.74±0.01,1.00±0.06 and 1.73±0.01,respectively.The expression levels of P53 mRNA in the above three groups were 1.94±0.01,0.81±0.03 and 1.92±0.01,respectively.The expression levels of Bax mRNA in the above three groups were 6.28±0.04,1.81±0.16,6.26±0.04,respectively.The trend of protein results was consistent with that of mRNA.The expression of HBV-DNA in the above three groups were(1032.68±2.96),(541.92±6.78)and(1035.44±1.86)pg·m L^(-1),respectively.HBsAg expression in the above three groups was(905.19±1.41),(246.23±7.04)and(902.56±4.74)U·m L^(-1),respectively.The expression of HBeAg in the above three groups was(45.28±0.06),(10.09±0.68)and(45.82±0.36)U·m L^(-1),respectively.The differences of the above indexes between transfection group and control group were statistically significant(all P<0.05).Conclusion Down-regulation of USP7 can reduce the expression of P53,decrease the expression of Bax,and reduce the replication of hepatitis B virus.