TGFβ1shRNA靶向抑制离体胎鼠肺成纤维细胞TGFfβ1基因表达

目的:探讨自行设计的TGFβ1shRNA对离体胎鼠肺成纤维细胞TGFβ1基因表达的干扰作用,为研究纤维化病变的基因治疗提供技术基础和依据.方法:原代培养胎鼠肺成纤维细胞,并建立细胞高氧损伤模型.针对大鼠TGFβ1基因mRNA序列,设计、合成携带3条TGFβ1shRNA绿色荧光蛋白融合表达质粒载体,并设阴性质粒组和空白组为对照,通过JetPEI包裹分别转染上述高氧损伤的胎鼠肺成纤维细胞.转染后24、48和72 h收集细胞,在荧光显微镜下观察干扰效果,采用实时荧光定量PCR检测TGFβ1基因表达情况,并计算干扰效率.结果:①成功培养胎鼠肺成纤维细胞,并建立细胞高氧损伤模型;②荧光显微镜下观察,可见转染后24、48和72 h TG-Fβ1shRNA质粒组细胞绿色荧光强度均明显弱于阴性质粒组细胞,空质粒载体组未产生绿色荧光;荧光定量PCR检测转染后胎鼠肺成纤维细胞TGFβ1 mRNA表达量,转染后24、48和72 hTGFβ1shRNA质粒组TGFβ1 mRNA表达量均显著低于阴性质粒组(P<0.01),其基因干扰效率则依次递减,分别为97.3%、96.9%和71.7%.结论:本研究证明自行设计的TGFβ1shRNA转染胎鼠肺成纤维细胞后24、48和72 h均能够高效干扰TGFβ1基因的表达,其基因干扰效率呈现一定的时间依赖性.

Inhibition of TGFβ1 gene expression by TGFβ1 short hairpin RNA on lung fibroblasts of embryonic rats in vitro

Aim:The effects of RNA interference on TGFβ1 gene in lung fibroblasts of embryonic rats in vitro through TGFβ1 short hairpin RNAs designed by myself were investigated in order to provide the foundation and evidence of a potential siRNA therapy of fibrotic diseases. Methods: Lung fibroblasts of embryonic rats were primary cultured and the model of hyperoxia induced lung fibroblasts injury were established.Three short hairpin RNAs targeting the TGFβ1 mRNA sequence and constructing the green fluorescence plasmid DNA of expressing TGFβ1 short hairpin RNAs were transfected into those cells model above mentioned. To determine the effect of these short hairpin RNAs,the positive plasmid vectors of TGFβ1 were transfected into these cells with the JetPEI, also used negative and null vectors were treated as above as controls. After 24,48 and 72 hours,the preliminary effect was evaluated with fluorescence microscope,and then fluorescent quantitation PCR was used to detect the expression of TGFβ1 gene at mRNA level. Moreover,calculated the efficiency of RNA interference. Results: ①Lung fibroblasts of embryonic rats were successfully cultured and the model of hyperoxia induced lung fibroblasts injury were established. ②Lung fibroblasts by the green fluorescence microscope were observed, fluorescence intensity decreased in the TGFβ1 short hairpin RNAs plasmid groups compared with the negative plasmid groups at 24,48 and 72 hours after transfection. Fluorescence was not observed in the null plasmid groups. The expression of TGFβ1 gene at mRNA level with fluorescent quantitation PCR was detected,the TGFβ1 gene expression was highly knocked down in the groups of TGFβ1 short hairpin RNAs plasmid compared with groups of negative plasmid at 24,48 and 72 hours after transfection (P<0.01 ), and their efficiency of RNA interference reduced by degree following times, were 97.3% ,96.9% and 71.7% respectively. Conclusion: The results suggested that the TGFβ1 short hairpin RNAs designed can interfere the expression of TGFβ1 gene efficiently at 24,48 and 72 hours after transfection and the efficiency of RNA interference reduced by degree following times.