靶向preC／C基因特异性RNA干扰对乙型肝炎病毒在肝癌细胞系中复制与表达的影响  

PreC/C gene-targeting RNA interference suppresses hepatitis B virus replication and expression in human hepatoma cells

作　　者：边中启[1] 刘霜[1] 刘明秋[2] 肖安[1] 焦晔[2] 严维耀[2] 郑兆鑫[2] 刘雪松（编辑）[1] 

机构地区：[1]解放军成都军区昆明总医院传染病中心,昆明650032 [2]复旦大学遗传所,昆明650032

出　　处：《中华医学杂志》2012年第11期768-772,共5页National Medical Journal of China

基　　金：国家自然科学基金（30972629）

摘　　要：目的探讨靶向乙型肝炎病毒（HBV）preC／C基因的小干扰RNA（siRNA）在人类肝癌细胞系Huh-7细胞和HepG2．2．15细胞中抗病毒基因治疗效果。方法根据GenBank中HBV（GenBank登录号U95551）基因组序列，设计合成靶向HBVpreC／C基因的3个长2l核苷酸（nt）的siRNA，设计1个对照的非同源长2Int的siRNA，分别克隆到pU6质粒中，构建3个shRNA表达载体pU6-C1，pU6-C2，pU6-C3和对照pU6-C4；为了检测siRNA功能建立报告基因系统，以pT-HBVl．3为模板，PCR合成目的基因preC／C，克隆到绿色荧光蛋白（EGFP）表达载体（pEGFP-N1）中构建携带EGFP报告基因的重组质粒pEGFP．preC／C（E-C）。将构建的3个shRNA表达载体与E-C共转染Huh-7细胞，或将该3个shRNA表达载体共转染HepG2．2．15细胞。首先，在Huh-7细胞中使用荧光显微镜观察及流式细胞仪检测EGFP融合表达细胞计数，评估该shRNA在转染后不同时间对EGFP融合报告基因表达的抑制效应；接着在HepG2．2．15细胞中，运用ELISA检测转染后24、48、72和96h细胞上清中HBsAg和HBeAg的表达量；免疫荧光技术检测转染后72h细胞内HBsAg和HBcAg的表达水平；实时荧光定量PCR（real-timePCR）进一步检测HBVmRNA转录产物cDNA的拷贝变化。结果发现siRNA表达质粒与E．C共转染Huh-7细胞后48h，与pEGFP-N1单质粒转染相比，pU6-C1，pU6-C2或pU6-C3共转染组使EGFP表达水平降低了80％，而对照pU6-CA或pU6共转染组无显著降低EGFP的表达，差异有统计学意义（P〈0．01）；免疫荧光技术检测发现HepG2．2．15细胞内HBsAg和HBcAg表达显著降低，real-timePCR发现pU6．C1、pU6-C2或pU6．C3共转染HepG2．2．15细胞后48h，使mRNA转录产物的cDNA含量分别降低了73．9％±1，2％（P=0．029）、48．2％±1．8％和35．8％±1．4％（P：0．037，0．040），而不论pU6-CA或pU6对照共转染组均不能降低cDNA含量，差异均有统计学意义（均P〈0．01）；pU[ Abstract ] Objective To explore the antiviral efficacy of small interfering RNAs （siRNAs）/ shRNA targeting preC/C of HBV in human hepatoma cells Huh-7 and HepG2. 2. 15 cells. Methods Three 21 nucleotide （nt）siRNAs for treating HBV preC/C gene were designed and synthesized according to theHBV genome in GenBank accession numbers （U95551）; simultaneously, one 21-nt-long non-homologous siRNA was also designed randomly for negative control. They were cloned into vector pU6 for constructing shRNA-expressing plasmids pU6-C1, pU6-C2, pU6-C3 and control pU6-C4. To assess the function of siRNAs, a reporter gene system was constructed. The HBV preC/C gene was synthesized by PCR with pT- HBV1.3 as the template. The preC/C gene was then inserted into the enhanced green fluorescent protein expression vector （ EGFP-N1 ） in order to construct the recombinant plasimid pEGFP-preC/C （ E-C）, which carries the EGFP reporter gene. The three shRNA-expressing plasmids-pU6-C1, pU6-C2, or pU6-C3-was each then cotransfected into Huh-7 cells along with either reporter gene expression vector E-C or the controls; or these three plasmids-pU6-C1, pU6-C2, or pU6-C3-was each cotransfected into HepG2. 2. 15 cells along with the controls. First, upon determination of the number of ceils exhibiting EGFP expression in Huh-7cells as detected by an BH-2 fluorescence microscope and FACS-440 flow cytometry at different times after cotransfection, the investigators evaluated the inhibitory efficiency of the three shRNA-expressing plasmids by an EGFP reporter system in cultured cells. Subsequently, the expression amount of HBsAg and HBeAg in HepG2. 2. 15 cell supernatant at 24, 48, 72 and 96 h post-cotransfection was detected by enzyme- linked immunosorbent assay （ELISA）. Immunofluorescence was used to detect the expression of HBsAg and HBcAg at 72 h post-cotransfection in HepG2. 2. 15 cells. The copy level of HBV mRNA transcripts cDNA in HepG2. 2. 15 cells was further investigated through quantitative real-time polymerase chain r

关 键 词：肝炎病毒 乙型 RNA干扰 基因疗法 

分 类 号：R735[医药卫生—肿瘤] R51[医药卫生—临床医学]