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HBV rtA181T突变对Huh7细胞自噬的影响
Effect of hepatitis B virus rtA181T mutation on autophagy of Huh7 hepatoma cells
文章发布日期:2016年09月28日  来源:  作者:时景仁,郭向华,王洁琳,等  点击次数:1469次  下载次数:315次

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【摘要】:目的探讨HBV逆转录酶区A181T(rtA181T)位点突变对Huh7细胞自噬的影响。方法Huh7细胞分别转染1.3mer HBV野生型质粒(WT组)、1.3mer HBV rtA181T突变型质粒(181T组)及1∶1共转WT和rtA181T突变型质粒(WT+181T组)。Western Blot、免疫荧光检测细胞内HBV表面蛋白(HBs)表达水平,ELISA检测细胞外HBsAg表达水平;Western Blot分析内源性自噬相关蛋白微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)及P62表达水平,Real-time PCR检测自噬相关基因Atg5 mRNA、Beclin1 mRNA的转录水平;同时转染绿色荧光蛋白LC3后,免疫荧光检测自噬颗粒。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。 结果免疫荧光观察181T组、WT组及WT+181T组细胞内均有HBs表达,Western Blot结果显示181T组细胞内的HBs表达高于WT组和WT+181T组。ELISA检测3组细胞培养上清的HBsAg水平差异有统计学意义(F=66.739,P<0001),其中181T组细胞外的HBsAg水平(0.111±0.056)远远低于WT组(3.157±0.490)和WT+181T组(2.240±0.797)(P值均<0001);WT组、181T组和WT+181T组均可见LC3-Ⅱ自噬颗粒聚集,181T组LC3-Ⅱ表达水平高于WT组和WT+181T组,而P62水平低于WT组和WT+181T组,差异均有统计学意义(P值均<0.05);181T组自噬相关基因Atg5 mRNA、Beclin1 mRNA转录水平均明显高于WT组和WT+181T组(P值均<0.05)。结论HBV rtA181T突变可导致HBsAg分泌障碍,并增加Huh7细胞的自噬水平。
【Abstract】:ObjectiveTo investigate the effect of reverse transcriptase A181T (rtA181T) mutation in hepatitis B virus (HBV) on autophagy of Huh7 hepatoma cells. MethodsHuh7 hepatoma cells were transfected with wild-type (WT) 1.3mer HBV plasmids (WT group), 1.3mer HBV rtA181T mutant plasmids (181T group), or WT and rtA181T mutant plasmids at a ratio of 1∶1 (WT+181T group). Western blot and immunofluorescence assay were performed to measure the expression of intracellular HBV surface protein (HBs), and ELISA was used to measure the expression of extracellular HBsAg. Western blot was used to measure the expression of endogenous autophagy-related proteins microtubule-associated light chain 3-II (LC3-Ⅱ) and P62, and Real time PCR was used to measure the mRNA expression of autophagy-related genes Atg5 and Beclin1. After transfection with green fluorescent protein-LC3, immunofluorescence assay was used to measure autophagy particles. An analysis of variance was used for comparison of continuous data between multiple groups and the LSD-t test was used for further comparison between any two groups. ResultsImmunofluorescence assay showed that there was expression of HBs in the 181T group, WT group, and WT+181T group, and the 181T group had significantly higher expression of intracellular HBs than the WT group and WT+181T group. The results of ELISA showed that there were significant differences in the level of HBsAg in cell culture supernatant between the three groups (F=66.739, P<0.001), and the 181T group had a significantly lower level of extracellular HBsAg than the WT group and WT+181T group (0.111±0.056 vs 3.157±0.490/2.240±0.797, both P<0.001). The WT group, 181T group, and WT+181T group all showed the aggregation of LC3-II autophagy particles; compared with the WT group and WT+181T group, the 181T group had significantly higher expression of LC3-II and significantly lower expression of P62 (both P<0.05). The 181T group hadsignificantly higher mRNA expression of autophagy-related genes Atg5 and Beclin1 than the WT group and WT+181T group (P<0.05). ConclusionHBV rtA181T mutation can cause reduced HBsAg secretion and increase autophagy level in Huh7 cells.
【关键字】:肝炎病毒,乙型; 突变; 自噬
【Key words】:hepatitis B virus; mutation; autophagy
【引证本文】:时景仁, 郭向华, 王洁琳, 等. HBV rtA181T突变对Huh7细胞自噬的影响[J]. 临床肝胆病杂志, 2016, 32(11): 2107-2111.

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