【作者】 边中启； 刘霜； 肖安； 刘明秋； 焦晔； 严维耀； 郑兆鑫；

【Author】 BIAN Zhong-qi~*,LIU Shuang,LIU Ming-qiu,XIAO An,JIAO Ye,YAN Wei-yao, ZHENG Zhao-xin.*Center for Infectious Diseases,CPLA Kunming General Hospital,Kunming 650032,P.R.China

【机构】 中国人民解放军成都军区昆明总医院传染病中心； 复旦大学遗传所；

【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HBV preC/C基因的3个长21核苷酸(nt)的siRNA,随机设计1个非同源对照21nt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-HBV1.3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24h,48h,72h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-time PCR)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-N 1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80%,而对照pU6-C4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-time PCR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-C4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(P<0.01);发现pU6-C1结果与显微镜观察/流式细胞仪细胞计数、ELISA和免疫荧光技术检测结果相吻合。抗病毒效果48h后作用明显,72h达到高峰。结论发现靶向preC/C基因的RNAi能够有效特异抗HBV在人类肝癌细胞系中复制与表达。RNAi可能成为抗重大传染病HBV/HCV/HIV有效的基因治疗技术。更多还原

【Abstract】 Object 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 the HBV 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-Cl,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 cells 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 24h,48h,72h and 96h post-cotransfection was detected by enzyme-linked immunosorbent assay(ELISA).Immunofluorescence was used to detect the expression of HBsAg and HBcAg at 72h 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 reaction(Real-time PCR).Results In comparison with single plasmid transfection pEGFP-N1 or E-C,fluorescence microscope examination and flow cytometry detection at 48 hours after cotransfection indicated that the expression of the reporter gene EGFP in cotransfected group Huh-7 cell involving pU6-C1,pU6-C2 or pU6-C3 resulted in an 80%reduction in EGFP signal relative to the controls(P <0.01).It was also found through immunofluorescence that the expression of HBsAg and HBcAg in HepG2.2.15 cells was reduced markedly(P < 0.01),that the copy level of HBV mRNA transcripts cDNA as detected at 48 hours after cotransfection by quantitative real-time PCR was reduced respectively by 73.9±1.2%(P=0.029;P < 0.05 ),48.2±1.8%and 35.8±1.4% (P=0.037,0.040;P < 0.05 ) relative to the control,that it conformed with that detected by fluorescence microscope/flow cytometry,ELISA,and immunofluorescence(P<0.01).Thereby further corroborating the antiviral efficacy of RNAi.The efficacy was obvious at 48h,reaching a peak at 72h.Conclusion For the first time it has been found that RNAi induced by siRNA/shRNA targeting HBV preC/C gene is effective and specific in inhibiting HBV replication and expression in human hepatoma cells Huh-7 and HepG2.2.15 cells.Our data suggest that RNAi may provide an effective, viable approach in gene therapy to treating major infectious diseases such as HBV/HCV/HIV infection.更多还原