Notch1受体在乙型肝炎病毒X基因介导的肾小管上皮细胞增殖、凋亡中的作用

摘要

目的 探讨乙型肝炎病毒X基因(HBx)转染人近端肾小管上皮细胞系HK-2细胞株后对其表达Notch1受体的影响,并观察Notch1受体在HBx介导的肾小管上皮细胞增殖、凋亡中的作用.方法 用分子克隆的方法构建pcDNA3.1/myc-HBx质粒及pcDNA3.1/myc-Notch1质粒,转染HK-2细胞建立转染株,并应用短发夹RNA(shRNA)干扰技术沉默Notch1基因表达.将细胞分为7组:①正常培养组；②转染HBx空载质粒组；③转染HBx质粒组；④转染HBx质粒+Notch1空载质粒组；⑤转染HBx质粒+Notch1质粒组；⑥转染HBx质粒+Notch1 shRNA空载质粒组；⑦转染HBx质粒+ Notch1 shRNA沉默质粒组.实时定量PCR和Western印迹法验证HBx转染成功,检测细胞中Notch1受体的变化,四甲基偶氮唑盐(MTT)检测各组细胞的生长抑制率,流式细胞术检测各组细胞凋亡情况.结果 HK-2细胞经转染HBx基因后可高表达HBx,Notch1 shRNA在HK-2细胞中的转染效率也高达70％,基因沉默有效；转染HBx基因组细胞Notch1水平明显高于正常培养组及转染HBx空载质粒组,且该组细胞凋亡率亦明显高于正常培养组及转染HBx空载质粒组,差异均有统计学意义(14.94％ ±0.32％比13.86％ ±0.18％、13.67％±0.54％,均P＜0.05).转染HBx质粒+Notch1质粒组细胞凋亡率明显低于HBx质粒+Notch1空载质粒组,差异有统计学意义(10.10％ ±0.26％比14.79％ ±0.02％,P＜0.05),其细胞增殖抑制率亦低于HBx质粒+Notch1空载质粒组.而转染HBx质粒+ Notch1 shRNA沉默质粒组细胞凋亡率及细胞增殖抑制率均高于HBx质粒+Notch1 shRNA空载质粒组(均P＜0.05).结论 构建pcDNA3.1/myc-HBx质粒并转染肾小管上皮细胞可成功制备乙型肝炎病毒细胞感染模型,由此导致的肾小管上皮细胞生长抑制及凋亡增加可能与Notch1受体异常活化有关.%Objective To explore the expression of Notchl receptor in renal tubular epithelial cells transfected with HBx gene and elucidate its role in cell apoptosis.Methods The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notch1 containing Notch1 gene was transiently transfected into HK-2 cells and shRNA technique employed for silencing Notch1.HK-2 cells were divided into 7 groups of normal culture,pcDNA3.1/myc,HBx,HBx + pcDNA3.1/myc,HBx + Notch1 gene,HBx + shRNA and HBx + Notch1 shRNA.Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of HBx and detect the expression of Notch1 receptor.Methyl thiazolyl tetrazolium (MTT) assay was used to observe the proliferation rate of HK-2 cells and flow cytometry to detect the apoptosis of HK-2 cells.Results HBx and Notch1 receptor were successfully expressed in HK-2 cells post-transfection.The transfection efficiency of shRNA was 70％.The expression of Notch1 receptor in pcDNA3.1/myc-HBx group was higher than that in the normal culture and pcDNA3.1/myc groups.And the apoptotic ratio was also higher than that of normal culture and pcDNA3.1/myc group.The difference was significant (14.94％ ± 0.32％ vs 13.86％ ± 0.18％,13.67％ ±0.54％,both P ＜ 0.05).The apoptotic ratio in the HBx + Notch1 gene group was lower than that in control group (10.10％ ±0.26％ vs 14.79％ ±0.02％,P ＜0.05).And the growth inhibition ratio of cells was also lower.But the apoptotic ratio and growth inhibition ratio were both higher in HBx + Notch1 shRNA group than those in HBx + shRNA group (both P ＜0.05).Conclusions HBx gene is successfully transfected into HK-2 cells.And its overexpression may induce apoptosis of HK-2 cells and growth inhibition by regulating Notch1 signal.