siRNA沉默HBx基因对肝癌细胞YKL-40蛋白表达影响的研究  

The Effects of Silencing HBx Gene by siRNA on the Expression of YKL-40 Protein in Hepatocellular Carcinoma Cell

作　　者：张永红[1] 贺兴鄂[1] 唐晓鹏[1] 王文龙[1] 罗开忠[1] 

机构地区：[1]中南大学湘雅二医院肝病研究所,长沙410011

出　　处：《中国医科大学学报》2010年第4期241-244,共4页Journal of China Medical University

基　　金：国家自然科学基金资助项目(30371402)

摘　　要：目的观察HepG2.2.15细胞中乙型肝炎病毒X(HBx)基因沉默对酿酒酵母YKL-40蛋白表达的影响。方法构建靶向HBx基因的siRNA真核表达载体pRNAT-HBx和阴性对照质粒,同时设空白对照组,采用Lipofectinamine 2000脂质体转染法转染人肝癌细胞系HepG2.2.15细胞。采用RT-PCR检测各组中HBx和YKL-40的mRNA水平,Western blot、细胞免疫荧光检测各组中HBx蛋白和YKL-40蛋白的表达。结果pRNAT-HBx明显抑制HepG2.2.15细胞中HBx基因的表达,同时YKL-40的mRNA和细胞内蛋白表达水平也随之相应下调。结论抑制HBx基因的表达能下调HepG2.2.15细胞中YKL-40蛋白的表达,提示HBx蛋白对YKL-40蛋白的表达具有一定的激活作用。Objective To observe the YKL-40 protein expression in HepG2.2.15 cells after hepatitis B virus X gene silence induced by siRNA.Methods Three short hairpin RNA interference plasmids targeting HBx gene were prepared as 3 groups：pRNAT-HBx（with interfering effect）,and pRNAT-Mock（negative control plasmid,without interfering effect）.Meanwhile,an empty control group was also designed.Transfection was performed using the Lipofectmine2000 liposome.Reverse transcription-polymerase chain reaction（RT-PCR）,Western blot and cell immunofluorescence were employed to detect the expression levels of HBx and YKL-40 gene and protein in HepG2.2.15 cells after transfection.Results The expression of HBx gene was significantly inhibited after transfection .At the same time,YKL-40 gene mRNA expression,the YKL-40 protein level in HepG2.2.15 were down-regulated in the pRNAT-HBx transfected group as compared with that in the pRNAT-Mock group.Conclusion Short hairpin RNA interference targeting HBx may inhibit the expression of YKL-40 gene in HepG2.2.15 cells.The results may indicate HBx protein could activate the YKL-40 protein expression.

关 键 词：小干扰RNA HEPG2.2.15细胞 乙型肝炎病毒X基因 YKL-40蛋白 

分 类 号：R3[医药卫生—基础医学]