Influence Of RNA Interference-induced Smad4 Gene Silencing On The Proliferation In Human Scar Fibroblast

Influence of RNA interference-induced Smad4 gene silencing on the proliferation in human scar fibroblastObjective:To investigate the effect of Smad4 gene silencing on the proliferation in human scar fibroblast.Methods:Two fragments of 19pb interference sequence for human Smad4 gene were designed by uesing Ambion RNAi online software. Synthetic oligonucleotide sequence of small hairpin (shRNA), and then cloned into the eukaryotic expression vector pSilencer TM 3.1-H1 neo vector, constructed recombinant plasmid. The plasmids were transfected into human phoroblast by liposome. Using RT-PCR, Western blot to detect Smad4 gene expression. The effect of Smad4 shRNA on the proliferation of human hyperplastic scar fibroblasts was assessed by MTT and Giemsa staining.Result:pSilencerTM 3.1-H1 neo-Smad4 expression vector were successfully constructed and identified by sequence analysis. Sequence analysis of inserted fragment revealed the same sequence as synthesized shRNA oligonucleotides. The results of RT-PCR and Western blotting show that transfection of pSilencerTM 3.1-H1 neo-Smad4 shRNA1 and shRNA2 can significantly down regulate the Smad4 mRNA and protein expression, especially shRNA1 have better marked effect(P<0.01). MTT results showed that Smad4 shRNA1 significantly inhibition of hypertrophic scar fibroblasts proliferation, and the proliferation activity decreased 40% compared with control cell (P<0.01). Giemsa staining of fibroblasts appeared distinct morphological changes such as formation of apoptotic bodies and cytoactive degrade, prominent shorter and fewer. Conclusion:The recombinant plasmid expressing the shRNA targeting Smad4 gene has been successfully constructed.Smad4 shRNA can significantly inhibit the proliferation of human hyperplastic scar fibroblasts.