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目的了解基于载体的siRNA对HepG2.2.15细胞中HBV的抑制作用,观察特异性siRNA载体对细胞的毒副作用及可能的非靶向效应,探讨新的抗病毒治疗手段。方法根据GenBank收录HepG2.2.15细胞系中HBV基因全序列,设计构建针对HBV的C基因区的3条siRNA的双链复合体,经退火连接入p-Silencer-Cmv4.1载体,连接产物转化JM109细胞,载体经聚丙烯酰胺凝胶电泳及测序鉴定后中量超纯提取质粒,定量后与转染试剂siPort XP-1转染HepG2.2.15细胞。免疫荧光及Western blot检测病毒蛋白HBsAg、HBeAg的表达;RT-PCR检测HBV RNA的表达变化;Real-time PCR定量检测HBV DNA拷贝变化。同时采用MTT法检测细胞的生长曲线与生长率。ELISA法检测IFN-α的表达变化。结果Western Blot结果显示在转染第3天p-C2对HBsAg、HBeAg的抑制率分别为(81.15±0.69)%、(88.12±0.92)%,免疫荧光显示同样结果。RT-PCR检测结果显示C基因特异的表达性siRNA可以显著抑制HBV的mRNA表达,对C区HBV mRNA的抑制达96.9%。定量PCR检测显示特异性siRNA使HBV DNA拷贝数降低102个数量级。而MTT检测表明该特异性siRNA表达载体不影响细胞的生长曲线与生长率,治疗组与对照组比较无差异。ELISA法检测表明特异性siRNA并不影响细胞干扰素的表达,p-C1、p-C2、p-C3组与未转染组、p-NC组比较表达均无差异。结论基于载体的特异性siRNA在体外可抑制HepG2.2.15细胞中HBV的复制和表达。该抑制作用是特异性的,同时对细胞无明显毒副作用。该作用为基因水平的裂解作用,不会出现耐药现象。
OBJECTIVE: To investigate the inhibitory effect of vector-based siRNA on HBV in HepG2.2.15 cells and to observe the toxicity and possible non-target effect of specific siRNA vector on cells and to explore new anti-virus therapy. Methods According to the full-length HBV gene sequence of HepG2.2.15 cell line in GenBank, three siRNA duplexes targeting to the C gene region of HBV were designed and annealed to the p-Silencer-Cmv4.1 vector, and the products were transformed into JM109 After the cells and the vector were identified by polyacrylamide gel electrophoresis and sequencing, the medium and the medium were extracted and the plasmid was transfected into HepG2.2.15 cells with the transfection reagent siPort XP-1. Immunofluorescence and Western blot were used to detect the expression of HBsAg and HBeAg. RT-PCR was used to detect the expression of HBV RNA. Real-time PCR was used to detect HBV DNA copy number. At the same time, the cell growth curve and growth rate were detected by MTT assay. The expression of IFN-α was detected by ELISA. Results The results of Western Blot showed that the inhibition rates of HBsAg and HBeAg by p-C2 on day 3 were (81.15 ± 0.69)% and (88.12 ± 0.92)%, respectively. Immunofluorescence showed the same result. The results of RT-PCR showed that C gene-specific siRNA could significantly inhibit the expression of HBV mRNA and inhibit the expression of HBV mRNA in C region by 96.9%. Quantitative PCR assays showed that specific siRNA reduced HBV DNA copy number by 102 orders of magnitude. The MTT test showed that the specific siRNA expression vector does not affect the cell growth curve and growth rate, the treatment group and the control group no difference. The results of ELISA showed that specific siRNA did not affect the expression of interferon. The expression of p-C1, p-C2 and p-C3 had no difference with those of untransfected and p-NC groups. Conclusion Specific vector based siRNA can inhibit HBV replication and expression in HepG2.2.15 cells in vitro. This inhibition is specific, with no significant toxic side effects on the cells. The role of gene level of the cleavage, there will be no resistance phenomenon.