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目的:评估以U6启动子作为启动序列(pSilencer2.0-U6)载体介导的小RNA干扰片段在培养细胞株中抑制HBV复制的效应.方法:将针对HBV基因组不同区域(S,X及C区)的核苷酸序列插入至pSilencer载体中,并将重组后的pSilencer质粒(分别为pS,pX及pC)载体转染入HepG2-N10细胞株(可稳定表达HBsAg、HBeAg及adw2亚型Dane颗粒)中.以ELISA法检测病毒抗原,以逆转录-聚合酶链反应法(reversetranscription-polymerasechainreaction,RT-PCR)检测病毒mRNA,并以荧光定量PCR法检测分泌入培养基的共价闭合环状DNA(covalentclosedcircularDNA,cccDNA).结果:质粒载体介导的RNA干扰能明显抑制培养基中HBsAg及HBeAg的表达.并且RT-PCR法检测显示病毒mRNA也被降解了,从而减少了蛋白表达及病毒逆转录复制的模板.荧光定量PCR法检测显示分泌入培养基的cccDNAs明显下降(HBVDNAlog10:pS:4.00±0.13;pC:4.08±0.10;pX:4.28±0.06;pN:5.05±0.07;HepG2-N10:4.74±0.06;HepG2:<2.70).结论:RNA干扰能抑制HBV基因表达及病毒复制,并且RNA干扰可能为HBV的治疗带来巨大的变化.
OBJECTIVES: To evaluate the effect of suppression of HBV replication in cultured cell lines using a small RNA interference fragment mediated by the U6 promoter as a promoter sequence (pSilencer2.0-U6). Methods: To target different regions of the HBV genome (S, X, and C The nucleotide sequence of the region was inserted into the pSilencer vector, and the recombinant pSilencer plasmids (pS, pX and pC, respectively) were transfected into the HepG2-N10 cell line (stable expression of HBsAg, HBeAg and adw2 subtype Dane. Viral antigens were detected by ELISA and viral mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The covalently closed loops secreted into the culture medium were detected by fluorescent quantitative PCR. DNA (covalently closed circular DNA, cccDNA). Results: Plasmid-mediated RNA interference can significantly inhibit the expression of HBsAg and HBeAg in the culture medium. RT-PCR assay showed that the viral mRNA was also degraded, thereby reducing protein expression and virus reversal. The replicated template was detected. Quantification of cccDNAs secreted into the culture medium was revealed by fluorescence quantitative PCR (HBVDNAlog10: pS: 4.00±0.13; pC: 4.08±0.10; pX: 4.28±0.06; pN: 5.05±0.07; HepG2-N10: 4.74±0.06; HepG2:<2.70). On: RNA interference can inhibit HBV viral gene expression and replication, and RNA interference could bring great changes to the treatment of HBV.