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）；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。
[ 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 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 pur-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 〈 O. O1 ）, 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）, 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.
出处
《中华医学杂志》
CAS
CSCD
北大核心
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）
