【摘要】 目的应用过氧化氢（H₂O₂）诱导人皮肤成纤维细胞衰老,建立体外细胞衰老模型。方法组织块贴壁法分离培养人皮肤成纤维细胞,免疫荧光法对获得的细胞进行鉴定,采用50μmol/L、100μmol/L、200μmol/L、400μmol/L的H₂O₂处理细胞1h、2h两个时间点,然后正常培养4d,应用细胞增殖实验,衰老相关的β-半乳糖苷酶（SA-β-gal）染色实验,确定H₂O₂氧化应激下的损伤力和细胞衰老率。结果镜下可见细胞呈梭形或多角形,结合免疫荧光结果,分离获得的细胞即为成纤维细胞。用50μmol/L、100μmol/L H₂O₂处理1h、2h后虽然活细胞较多,但是SA-β-gal染色阳性率较低。200μmol/L、400μmol/L H₂O₂处理2h组SA-β-gal染色阳性率较高,但细胞量较少给以后实验带来较多困难。200μmol/L、400μmol/L H₂O₂处理1h组细胞数量,SA-β-gal染色阳性率最高且二者间无明显差异。结论选取200μmol/L H₂O₂处理1h作为建立衰老细胞模型。更多还原

【Abstract】 Objective To induce human dermal fibroblasts senescence by hydrogen peroxide and establish a model of premature senescence in vitro. Methods The human dermal fibroblasts were isolated and cultured by tissue patch method, immunofluorescence staining was conducted to identify the cells. The cells were treated with 50μmol/L,100μmol/L, 200μmol/L, 400μmol/LH₂O₂ for 1 h and 2 h. 4 days later, cell proliferation assay and SA-β-gal staining were used to determine the damage capacity and the percentage of senescence under oxidative stress. Results The cells were observed with spindle or polygonal shape, the fibroblasts were isolated successfully. The positive ratio of SA-β-gal staining cells was less although more living cells were treated with 50μMol/L,100μMol/L H₂O₂ for 1 h and 2 h, but higher treated with 200μmol/L or 400μmol/L H₂O₂. The number of cells or SA-β-gal positive ratio were the highest for 200 mol/L and 400 mol/L H₂O₂ for 1 h, with no significant difference between 200 mol/L and 400 mol/L H₂O₂. Conclusion The treatment with 200μmol/L H₂O₂ for 1 h was selected as the suitable senescence model.更多还原