HBV X基因转染L02细胞及其蛋白产物对CⅡTA和HLA-DR表达的影响

目的探讨脂质体和电穿孔法在HBV X基因转染L02细胞中的差别及X基因蛋白产物对CⅡTA和HLA-DR表达的影响。方法构建HBV X基因重组表达质粒,通过电穿孔和脂质体法转染人正常肝细胞L02细胞,以绿色荧光蛋白(enhance green fluorescent protein,EGFP)表达水平评价转染效率,流式细胞技术检测L02细胞HBV X蛋白的表达水平。RT-PCR、流式细胞技术及western-blot分析L02细胞、转染空载体以及转染HBV X基因重组表达质粒的L02细胞CⅡTA和HLA-DR的表达差别。结果采用阳离子脂质体Lipofectamine 2000的转染效率为(6.4±3.5)%,显著低于电穿孔法(41.46±5.8)%,P<0.01。脂质体法转染的L02细胞HBV X蛋白表达水平为(5.1±3.9)%,显著低于电穿孔方法(37.5±4.1)%表达,P<0.01。转染了HBV X基因重组表达质粒的L02细胞检测到CⅡTA和HLA-DR mRNA和蛋白表达,而L02细胞本身及转染空载体的L02细胞未检测到CⅡTA和HLA-DR的表达。结论 HBV X基因真核表达载体pHBx-IRES2-EGFP通过电穿孔法转染L02细胞较脂质体法有明显较高的转染效率和更高的目的基因的表达,HBV X基因诱导了L02细胞中CⅡTA和HLA-DR的表达。

Transfection of HBV X gene into human liver cell line L02 and its effect on CIITA and HLA-DR expression

Objective To investigate the efficiency difference between lipofection and electroporation for HBV X gene transfection and the effects of HBV X protein(HBx) on CIITA and HLA-DR expression in human liver cell line L02.Methods Recombinant expression plasmid pHBx-IRES2-EGFP was constructed and transferred into normal human liver cell line L02 with Lipofectamine 2000 and electroporation respectively.The transfection efficiency was evaluated by the expression of enhanced green fluorescent protein(EGFP) under a fluorescent microscope.Flow cytometry was used to detect HBx protein expression.RT-PCR,flow cytometry and Western blot analysis were used to determine HLA-DR and CIITA expression in L02 cells,L02 cells transfected with pIRES2-EGFP,and L02 cells transfected with pHBx-IRES2-EGFP respectively.Results The transfection efficiency with Lipofectamine 2000 was(6.4±3.5)%,significantly lower than that with electroporation (41.46±5.8)%,P<0.01].The HBx expression in the L02 cells transfected by Lipofectamine 2000 was significantly lower than that in the L02 cells transfected by electroporation (5.1±3.9)% vs(37.5±4.1)%,P<0.01].The CIITA and HLA-DR expression was only detected in the L02 cells transfected with pHBx-IRES2-EGFP.Conclusion Using recombinant expression plasmid pHBx-IRES2-EGFP,HBV X gene transfection mediated by electroporation achieves a higher efficiency and a higher HBx expression level in L02 cell lines than that mediated by Lipofectamine 2000.HBx induces CIITA and HLA-DR expression in L02 cells.