特异性shRNA抑制小鼠L929成纤维细胞Smad3基因表达的研究

Objective To design and produce specific shRNAs and detect their inhibitive effects on Smad3 expression. Methods According to Smad3mRNA, three shRNAs that encoded different subsequences were designed. The nucleotides of start site for shRNA1, shRNA2 and shRNA3 were 495, 562 and 1473 and the contents of GC were 42.1%, 52.6% and 52.6%. Three shRNAs were cloned into pGenesil1.1 plasmid. The control group (cells that not been transfected), the negative control group (cells that been transfected by the scrambled shRNA plasmid), the pGenesil1.1 Smad3-1, pGenesil1.1 Smad3-2 and pGenesil1.1 Smad3-3 groups were set up. Real time RCR and Western blot were conducted to detect the expression of Smad3 after 48h and 72 h of the transfection. Results Three shRNAs all inhabited the expression of Smad3 in different levels after 48h and 72 h of the transfection. It was noted that shRNA2 and shRNA3 had high efficiency. Conclusion The Smad3 expression of mice L929 fibroblast cells in vitro can be depressed by specific shRNA. It seems possible to provide an advanced research for the treatment of pulmonary fibrosis.