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HBs Ag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HBs Ag水平差异有统计学意义(F=66.739,P<0.001),其中181T组细胞外的HBs Ag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HBs Ag分泌障碍,并增加Huh7细胞的自噬水平。
Objective To investigate the effect of reverse transcriptase A181T（ rtA181T） mutation in hepatitis B virus（ HBV） on autophagy of Huh7 hepatoma cells. Methods Huh7 hepatoma cells were transfected with wild- type（ WT） 1. 3mer HBV plasmids（ WT group）,1. 3mer HBV rt A181 T mutant plasmids（ 181 T group）,or WT and rt A181 T mutant plasmids at a ratio of 1∶ 1（ WT ＋ 181 T 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 HBs Ag. 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. Results Immunofluorescence assay showed that there was expression of HBs in the 181 T group,WT group,and WT ＋ 181 T group,and the 181 T group had significantly higher expression of intracellular HBs than the WT group and WT ＋ 181 T group. The results of ELISA showed that there were significant differences in the level of HBs Ag in cell culture supernatant between the three groups（ F = 66. 739,P〈0. 001）,and the 181 T group had a significantly lower level of extracellular HBs Ag than the WT group and WT ＋ 181 T group（ 0. 111 ± 0. 056 vs 3. 157 ± 0. 490 /2. 240 ± 0. 797,both P〈0. 001）. The WT group,181 T group,and WT ＋ 181 T group all showed the aggregation of LC3- II autophagy particles; compared with the WT group and WT ＋ 181 T group,the181 T group had significantly higher expression of LC3- II and significantly lower expression of P62（ both P〈0. 05）. The
出处
《临床肝胆病杂志》
CAS
2016年第11期2107-2111,共5页
Journal of Clinical Hepatology
基金
国家自然科学基金(81361120401)
首都卫生发展科研专项项目(首发2014-1-1151)
肿瘤侵袭和转移机制研究北京市重点实验室开放课题(2015ZLQX05)
关键词
肝炎病毒
乙型
突变
自噬
hepatitis B virus
mutation
autophagy
