摘要
目的建立抑制p53基因表达的人皮肤成纤维细胞（HSF），探讨抑制p53表达对HSF衰老的影响。方法脂质体转染法将针对p53的shRNA的真核表达质粒pGCsi—p53导入HSF中，经G418筛选抗性克隆，扩大培养，建立稳定转染克隆细胞系。用RT—PCR、实时荧光定量PCR和蛋白印迹法分析目的基因及其蛋白表达。通过SA β—gal染色法检测细胞衰老，MTT法检测细胞增殖能力。结果成功建立RNA干扰抑制p53表达的HSF细胞系，转染细胞中p53基因及蛋白水平明显下调（P〈0．01）。sAB—gal染色结果显示，转染组衰老细胞百分比（13．47％±1．01％）与对照组（18．10％±0．66％）相比降低（P〈0．05），MTF检测结果显示，转染组细胞增殖能力较对照组加快（P〈0．05）。结论抑制p53表达能够延迟HSF的衰老，促进细胞增殖。
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 SAβ-gal staining, and cell proliferation by methyl thiazolyl tetrazolium （MTY） 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 （0.09：1：0.03 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 MrFF assay showed, the proliferation was accelerated in transfeeted HSFs compared with untransfeeted HSFs （P 〈 0.05）. Conclusions The repression of p53 expression decelerates the senescence in HSFs, but promotes the proliferation of HSFs.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2012年第11期799-802,共4页
Chinese Journal of Dermatology
基金
国家自然科学基金（30671894）
南京市卫生局青年科技人才启动项目（QYK10135）
