摘要
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 explore the siRNA as a new antiviral therapy, evaluate the inhibition effect of siRNA based on vector on the HBV of HepG2.2.15 cell,and observe the side effect and toxicity of siRNA vector on cells and the off - target effect of siRNA. Methods Three pairs of siRNA duplexes targeting HBV C gene were designed as double strands,and the duplex were annealed and ligated into the p - Silencer- Cmv 4.1 -hygro vector. The ligation products were used to transform JM109 cells. The clones with shRNA were obtained,and the vectors were purified. After the initial identification of the vector with agarose gel and the size of the inserted sequence got examined by native polyacrylamide gel electrophoresis, furthermore the sequencing was further carried out. The recombinant plasmids were purified with ultrapure Midipreps DNA Purification System. Then HepG2.2.15 cells were transfected with the plasmid mixed with siPort XP - 1. The expression of HBsAg and HBeAg were detected by immunofluoreseence and Western blot, and the HBV RNA was investigated by RT- PCR. Furthermore the real - time quantitive PCR was carried out to detect the changes of HBV DNA. In order to evaluate the toxicity of the shRNA, MTT was used to examine the growth rate and curve of cells. The ELISA was performed to detect the changes of interferon - α（ IFN - α）. Results The Western blot showed that the HBsAg and HBeAg protein were suppressed with （81.15 ± 0.69 ）%, （88.12 ± 0.92 ）% respectively by vector p - C2 on the third day of post - transfeetion. It had the similar result indicated by immunofluoreseenee. And the RT - PCR showed that the specific siRNA targeting HBV C gene could markedly suppress the expression of HBV mRNA and the HBV C gene mRNA was inhibited with 96.9%. The real - time quantitive PCR showed that the specific functional siRNA could markedly suppress HBV DNA copy with two orders of magnitude ,while the siRNA vector had no effect on the growth of cell showed by MTT detection. Compared with the non - transfected group
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2008年第22期1750-1753,共4页
Journal of Applied Clinical Pediatrics
