Twist1在人皮肤成纤维细胞中的表达及其对细胞增殖的影响

目的探讨Twist1对人皮肤成纤维细胞增殖的影响及其可能机制。方法组织块贴壁法提取人皮肤成纤维细胞(HSFs),通过连续传代建立人皮肤成纤维细胞的复制性衰老模型。将传代的HSFs分为年轻组(P3~7)、衰老组(P20~25),通过β-半乳糖核苷酶染色法比较衰老染色率、EdU检测细胞增殖率的变化以及Western blot法检测衰老相关蛋白p21表达的差异来验证复制性衰老模型是否成功建立。通过Western blot法检测Twist1分别在年轻组与衰老组的表达情况。接着在年轻组中,通过转染Si-Twist1序列,使Twist1的表达下降,EdU检测HSFs增殖率的变化。结果相比于年轻组,衰老组HSFs的SA-β-gal染色率明显增加,细胞增殖率明显下降,衰老相关蛋白p21的表达明显增加,差异均有统计学意义(P<0.05)。以上实验结果表明,已成功建立了HSFs的复制性衰老模型。另外Western blot发现Twist1蛋白在年轻组HSFs中的表达水平显著高于衰老组(P<0.05),EdU提示在Si-Twist1组比NC组的细胞增殖率明显下降(P<0.05)。结论 Twist1在衰老组HSFs中表达明显下调,在体外抑制Twist1的表达可引起HSFs的增殖率的下降,这为探讨Twist1与皮肤成纤维细胞衰老的相关研究提供了依据。

Expression of Twist1 in Human Skin Fibroblasts and Its Effects on Cell Proliferation

Objective To study the effect of Twist1 on proliferation of human skin fibroblasts(HSFs),and the possible mechanism.Methods HSFs were extracted by tissue block adherence method and a replicative senescence model of HSFs was established by serial passages.Subsequently,HSFs were divided into young group(P3~7)and aged group(P20~25).SA-β-gal staining was used to test the senescence staining rate,the cell proliferation rate was detected by EdU,and the expression of aging-related protein p21 was measured by Western blot.These results determined whether the replicative senescence model was successfully established.Western blot was used to detect the expression of Twist1 in young and aged group,respectively.Then,in young group,the expression of Twist1 was decreased by transfection of Si-Twist1,and the change of the cell proliferation rate was detected by EdU.Results Compared with young group,the percentage of positive cells for SA-β-gal staining in aged group was significantly higher,the cell proliferation rate was obviously lower,and the expression of aging-related protein p21 was notably higher(P<0.05).These experimental results showed that the replicative senescence model of HSFs was successfully established.In addition,Western blot was used to detect the Twist1 protein,which showed that the expression level of Twist1 protein in young group was evidently higher than that in passage-aged group(P<0.05).EdU result showed that the cell proliferation rate was lower in Si-Twist1 group than in NC group(P<0.05).Conclusion The expression of Twist1 in HSFs in aged group is distinctly down-regulated.Inhibition of Twist1 expression in vitro can significantly inhibit the proliferation of HSFs,which may provide a basis for the study of the relationship between Twist1 and senescence of skin fibroblasts.