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 quantitative 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.

Key words: HSF