D-半乳糖诱导小鼠原代皮肤成纤维细胞衰老模型的建立

Establishment of D-galactose induced primary skin fibroblasts aging model in mice

目的构建D-半乳糖诱导小鼠皮肤成纤维细胞(MSF)衰老模型。方法取5～7 d昆明乳鼠背部皮肤,采用Ⅰ型胶原酶消化法分离获得MSF,通过形态学观察及Vimentin免疫组织化学法鉴定MSF细胞。分别用不同浓度D-半乳糖诱导MSF细胞0、24、48 h,采用β-半乳糖苷酶衰老染色、逆转录-聚合酶链反应(RT-PCR)检测衰老相关基因p53、细胞免疫荧光法分析Sirt1基因表达水平,并分析细胞衰老情况。结果光学显微镜下细胞呈多突的梭形或星形的扁平细胞,Vimentin蛋白免疫组织化学分析呈阳性。β-半乳糖苷酶衰老染色实验结果显示,不同浓度D-半乳糖诱导后染色阳性细胞数量较对照组显著增多,差异有统计学意义(P<0.01)。在D-半乳糖(20 g/L)诱导24、48 h后,与对照组比较,衰老MSF p53基因表达水平显著升高,差异有统计学意义(P<0.01);细胞免疫荧光结果显示,D-半乳糖诱导48 h后Sirt1蛋白表达水平明显降低。结论通过Ⅰ型胶原酶消化法及D-半乳糖诱导法成功建立了衰老模型。

Objective To establish an aging model of mice skin fibroblasts （MSF） induced by D-galactose. Methods The skin on the back of 5-7 d mice was obtained. And the MSF were obtained by type I collagenase digestion. For identified MSF, morphology observation and cell immunohistochemistry was detected. After MSF treatment with D-galactose for 0,24,48 h, β-galactosidase aging staining, reverse transcription polymerase chain reaction （RT-PCR） of senescence related gene p53 and cellular immunofluorescence of Sirtl were detected for analysis of cell senescence. Results The cells obtained from type I collagenase digestion were showed in spindle-shaped or star-shaped fiat cell with protrusions under an optical microscope. And Vimentin protein was positive staining in the immunohistochemical analysis which suggested that the primary cultured cells were MSF. β-galactosidase assay- in MSF showed that the number of positive cells in MSF with different concetration of D-galac- tose were significant increased when compared with those of control group in 24 h and 48 h, there were Statistical significance （P〈0.01）. After induction by D-galactose （20 g/L）24 h and 48 h, the relative mRNA expressions of p53 in MSF were higher than that of control group, there were Statistical significance （P〈0.01）. The protein of Sirtl in MSF induced by D-galactose were lower than that of control group. Conclusion D-galactose induced primary skin fibroblasts aging model in mice is established by type I collagenase digestion and D- galactose induction in this study.