肝纤维化相关细胞因子双重RNA干扰质粒的构建及转染肝星状细胞研究

目的构建以大鼠CTGF和TIMP-1基因为靶点的双重RNA干扰表达载体质粒,以检测其转染肝星状细胞的效率.方法筛选出对CTGF和TIMP-1基因最有效的RNA干扰靶位,各设计1对含有短发夹结构的RNA 干扰靶点序列,分别克隆到质粒载体psiRNA-DUO-GFPzeo,构建含目的靶基因片段的重组质粒载体psiRNA-GFP-CT-GF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com（含有CTGF和TIMP-1）,进行酶切和测序鉴定.将构建成功的重组质粒psiRNA-GFP-CTGF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com转染大鼠肝星状细胞,在荧光显微镜下观察质粒转染情况,用流式细胞仪检测转染效率.结果酶切与测序结果提示重组质粒 psiRNA-GFP-CTGF、psiR-NA-GFP-TIMP-1和psiRNA-GFP-Com成功构建;成功将重组质粒psiRNA-GFP-CTGF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com转染肝星状细胞,在24小时,空质粒组、CTGF组、TIMP-1组和CTGF ＋TIMP-1组转染效率分别为15±2%、13±1%、15±1%和14±2%,均低于质粒psiRNA-GFP-Com转染组（Com组,20±2%,P〈0.05）;在48小时,空质粒组、CTGF组、TIMP-1组和CTGF＋TIMP-1组转染效率分别为10±2%、9±1%、8±2%和10±1%,均低于Com组的15±2%（P〈0.05）.结论成功构建靶向大鼠CTGF和TIMP-1最有效的RNA干扰靶位的双重RNA干扰表达质粒,能转染至肝星状细胞,并且psiRNA-GFP-Com转染效率最高.

Construction and transfection of hepatic fibrosis-related cytokines double RNA interference recombinant plasmids into hepatic stellate cells in vitro

Objective To construct double RNA interference recombinant plasmid targeting at genes of rat connective tissue growth factor（CTGF） and tissue inhibitor of metalloproteinase I（TIMP-1）. Then,the recombinant plasmids would be transfected into hepatic stellate cells（HSC）. Methods According to the most effective RNA interference sequences of rat CTGF gene and rat TIMP-1 gene screened out in the previous experiments,three pairs of CTGF and TIMP-lgene-specific shRNA were designed and synthesized. After primer annealing,they were inserted into plamid psiRNA-DUO-GFPzeo and named as psiRNA-GFP-CTGF,psiRNA-GFP-TIMP-1 and psiRNA-GFP-Com （with CTGF and TIMP-1）,respectively. The three recombinant plasmids were confirmed by restriction enzyme digestion and sequencing,and the three recombinant plasmids were transfected into hepatic stellate cells by liposomes 2000. Fluorescence microscope and flow cytometry were employed to meaure the plasmid transfeetion efficiency. Results The restriction enzyme digestion and sequencing showed that three shRNA recombinant plasmids were constructed and three shRNA recombinant plasmids were transfected into HSC successfully;At 24 hours after transfection,the transfection rates in empty plasmid group,CTGF group,TIMP-lgroup and CTGF plus TIMP- lgroup were 15±2%,13±1%,15±1% and 14±2%,respectively,much lower than that in psiRNA-GFP-Com group （20±2%,P〈0.05）;At 48 hours after transfection, the transfeetion rates in empty plasmid group,CTGF group , TIMP -1 group and CTGF plus TIMP-lgroup were 10±2%,9±1%,8±2%and 10±1%,respectively, much lower than that in psiRNA-GFP-Com group （15±2%,P〈0.05）. Conclusion Three shRNA recombinant plasmids are constructed successfully and can be transfeeted into HSCs,and the transfection efficiency in double RNA interference recombinant plasmid psiRNA-GFP-Com is high.