Influence of RNA interference-induced repression of p53 expression on senescence in human skin fibroblasts / 中华皮肤科杂志
Chinese Journal of Dermatology
; (12): 799-802, 2012.
Article
de Zh
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| ID: wpr-430393
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ABSTRACT
Objective To establish a cell line with repressed expression of p53 by transfecting a plasmid construct expressing short hairpin RNA(shRNA)targeting p53 into human skin fibroblasts(HSFs),and to evaluate the effect of repression of p53 expression on the senescence in HSFs.Methods The eukaryotic expressing plasmid pGCsi-p53 containing shRNA targeting p53 gene was transfected into HSFs with lipofectamine.Subsequently,the cells were selected by G418,and resistant cell clones were chosen and expanded.Reverse transcription-PCR and real time fluorescence-based quanitative PCR were performed to determine the expression of p53 gene,and Western blot to detect the expression of p53 protein in HSFs.The senescence in HSFs was evaluated by SA β-gal staining,and cell proliferation by methyl thiazolyl tetrazolium(MTT)assay.Results A HSF clone with repressed expression of p53 was established successfully.The expressions of p53 mRNA and protein were downregulated in transfected HSFs compared with untransfected HSFs(0.09 ± 0.03 vs.0.32 ± 0.04,0.11 ± 0.04 vs.0.84 ± 0.05,both P < 0.01).The percentage of senescent cells was 13.47% ± 1.01% in the transfected HSFs,significantly lower than that in untransfected HSFs(18.10% ± 0.66%,P < 0.05).As MTT assay showed,the proliferation was accelerated in transfected HSFs compared with untransfected HSFs(P < 0.05).Conclusions The repression of p53 expression decelerates the senescence in HSFs,but promotes the proliferation of HSFs.
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WPRIM
langue:
Zh
Texte intégral:
Chinese Journal of Dermatology
Année:
2012
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Article