| Objective:To investigate the effect on proliferation and apoptosis by siRNA which silences the expression of the TGF-βRⅡof human hyperplastic scar fibroblast(HSFb)in vitro.Methods:The small hairpin RNA(shRNA)expression vector pSilencerTM 3.1-H1-TGF-βRⅡshRNA1,2,3 were synthesized which according to 3 different target sequences of TGF-βⅡreceptor gene by recombinant DNA technology.The plasmids were transfected into human phoroblast by lipidosome approach and the cell clones which suppress the expression of TGF-βⅡreceptor were selected by G418.Effect of TGF-βRⅡsiRNA on the proliferation of human hyperplastic scar fibroblasts was assessed by MTT assay and the fibroblast apoptosis induced by TGF-βRⅡsiRNA were observed through Giemsa staining, flow cytometry(FCM)and 1.5%agarose gel electrophoresis.ResultIt was verified by partial nueleotide sequencing and restriction endonuelease digestion that the constructed eukaryotie vector expressing shRNA of TGF-βRⅡreceptor was correct,shRNA2 in pSilencerTM3.1-H1-TGF-βRⅡcells knocked down the expression of TGF-βⅡreceptor mRNA and protein dramatically compared with untransfected and control cells.After modulation of hypertrophic scar fibroblasts for 24,48 and 72 hours with pSilencerTM3.1-H1-TGF-βRⅡshRNA2,fibroblasts take on distinct morphological changes, including nuclear condenstation and digestion,membrane blebbing, and formation of apoptotic bodies,apoptotic percentage of human hypertrophic scar fibroblasts induced by pSilencerTM 3.1-H1-TGF-βRⅡshRNA for 24,48 and 72 hours was 18.4%. Apoptotic DNA ladder can be detected and the highest DNA levels were obtained at 48 hours.ConclusionThe shRNA expression vector pSilencerTM3.1-H1-TGF-βRⅡshRNA1,2,3 were synthesized and the cell clones which can efficiently suppress the expression of TGF-βⅡreceptor were selected.TGF-βRⅡsiRNA can inhibit proliferation of human hyperplastic scar fibroblasts obviously.TGF-βRⅡsiRNA can induces the apoptosis of human hypertrophic scar fibroblasts.
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