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,设计1个对照的非同源长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24、48、72和96 h细胞上清中HBsAg和HBeAg的表达量；免疫荧光技术检测转染后72 h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HBV mRNA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48 h,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 -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48 h,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(均P＜0.01)；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48 h后作用明显,72 h达到高峰.结论 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的复制与表达.RNAi可能成为抗重大传染病HBV/HCV/HIV有效的基因治疗技术.

abstractsObjective 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 gere 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 pTHBV1.3 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 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 24,48,72 and 96 h post-cotransfection was detected by enzymelinked 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 reaction ( realtime 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 cotansfection by quantitative real-time PCR was reduced respectively by 73.9％ ± 1.2 ％ ( P =0.029 ),48.2％ ± 1.8％ and 35.8％ ± 1.4％ ( P =0.037,0.040) 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 48 h,reaching a peak at 72 h.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.