乙肝病毒X基因参与肝细胞脂肪变性的作用及机制的初步研究  

Preliminary study of the effect and mechanism of hepatitis B virus X gene in hepatic steatosis

作　　者：张琴[1] 彭俊[2] 沈薇[1] 

机构地区：[1]重庆医科大学附属第二医院消化内科,重庆400010 [2]凉山州第一人民医院妇产科,四川西昌615000

出　　处：《第三军医大学学报》2011年第23期2466-2470,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30871160);重庆市自然科学基金(CSTC2008BB5404);重庆市医学科技计划项目(2008-2-401)~~

摘　　要：目的探讨沉默HBx基因对HepG2.2.15细胞脂质代谢相关基因表达的影响。方法设立空白对照组(HepG2.2.15细胞)、阴性对照组(HepG2.2.15细胞转染阴性HK质粒)、shHBx转染组(HepG2.2.15细胞转染shHBx质粒)和HepG2组(HepG2细胞)。构建针对HBx基因的shHBx质粒,转染HepG2.2.15细胞,荧光显微镜及Western blot检测转染质粒24～96 h绿色荧光蛋白和HBx蛋白的表达以确定质粒的最佳干扰时间,根据结果予油酸钠刺激细胞,甘油三酯(TG)检测细胞脂肪变程度,RT-PCR检测固醇调节元件结合蛋白(sterol regulatory element binding protein-1c,SREBP-1c)mRNA的表达,Western blot检测肝X受体α(liver x receptor alpha,LXRα)及脂肪酸合成酶(fatty acid synthase,FAS)蛋白的表达。结果成功构建shHBx质粒并转染HepG2.2.15细胞;与空白对照组和阴性对照组比较,shHBx转染组HBx蛋白表达明显下降,于转染后48～72 h表达最低[(0.337±0.042)vs(0.577±0.032),(0.333±0.032)vs(0.654±0.049),P<0.01],差异有统计学意义;随着油酸钠处理时间延长,各组TG含量、SREBP-1c mRNA水平、LXRα和FAS蛋白表达均逐渐增加,与空白对照组和阴性对照组比较,同一时间点,shHBx转染组和HepG2组中表达较低[TG:(26.51±1.98)μg/mgvs(37.16±6.32)μg/mg;SREBP-1c:0.418±0.051 vs 0.516±0.037;LXRα:0.463±0.047 vs 0.630±0.026;FAS:0.463±0.047 vs 0.630±0.017,P<0.01],差异有统计学意义,而shHBx转染组与HepG2组相比水平近似,差异无统计学意义(P>0.05)。结论 HBx在HepG2.2.15细胞脂变中具有重要作用,可能通过调节LXRα、FAS、SREBP-1c表达影响肝细胞脂代谢。Objective To investigate the effect of hepatitis B virus X（HBx） gene silencing on expression of lipid metabolism related genes in HepG2.2.15 cells.Methods Cells were divided into a blank control group（HepG2.2.15 cells）,a negative control group（HepG2.2.15 cells transfected with negative HK plasmids）,a shHBx group（HepG2.2.15 cells transfected with shHBx plasmids） and a HepG2 group（HepG2 cells）.shHBx plasmid was constructed and transfected into HepG2.2.15 cells using PolyJetTM reagent.Expressions of green fluorescent protein and HBx protein were examined under fluorescence microscope and by Western blotting after 24 h-96 h transfection to identify a best interference time.Then cells were treated with sodium oleate for 24 h and 48 h,respectively.Degree of hepatic steatosis was determined by triglyceride（TG） test.mRNA expression of sterol regulatory element binding protein-1c（SREBP-1c） was detected by reverse transcription polymerase chain reaction（RT-PCR）,and protein expressions of liver X receptor alpha（LXRα） and fatty acid synthase（FAS） were detected by Western blotting.Results shHBx plasmid was successfully constructed and transfected into HepG2.2.15 cells.Compared with the blank control group and negative control group,HBx protein level decreased significantly in the shHBx group.The lowest level was detected at 48 h-72 h after transfection（0.337±0.042 vs 0.577±0.032,0.333±0.032 vs 0.654±0.049,P〈0.01）.After cells were stimulated with sodium oleate,the TG content,SREBP-1c mRNA level and LXRα and FAS protein levels increased as the time extended,and they decreased significantly in the shHBx group and HepG2 group as compared with the blank control group and negative control group（TG： 26.51±1.98 μg/mg vs 37.16±6.32 μg/mg;SREBP-1c： 0.418±0.051 vs 0.516±0.037;LXRα： 0.463±0.047 vs 0.630±0.026;FAS： 0.463±0.047 vs 0.630±0.017,P〈0.01）.There was no significant difference between the shHBx group and the HepG2 group（P〈0.05）.Conclusion HBx plays

关 键 词：乙肝病毒X基因 肝细胞脂肪变性 质粒 RNA干扰 

分 类 号：R575.02[医药卫生—消化系统]