p21小干扰RNA对低浓度过氧化氢诱导的黑素细胞提前衰老和黑素传递的影响

Effect of p21 siRNA on premature senescence and melanosome transfer induced by a low concentration hydrogen peroxide in human melanocytes

目的:探讨小干扰RNA(siRNA)抑制p21表达对低浓度过氧化氢(H2O2)诱导的黑素细胞提前衰老的影响,以及黑素细胞提前衰老与黑素传递功能间的关系。方法:p21 siRNA序列转染黑素细胞,采用荧光显微镜、实时荧光定量聚合酶链式反应(qRT-PCR)及western blot检测抑制效率。将正常人黑素细胞分为对照组(阴性siRNA转染处理)、p21 siRNA组(p21 siRNA转染处理)、阴性siRNA+H2O2组(阴性siRNA转染24 h后加入400μmol/L H2O2处理)及p21 siRNA+H2O2组(p21 siRNA转染24 h后加入400μmol/L H2O2处理)。采用β-半乳糖苷酶染色检测细胞衰老;EdU细胞增殖检测细胞增殖能力;western blot检测各组p21和树突调节蛋白[RhoA、Rac1及细胞分裂周期蛋白42(Cdc42)]表达;免疫荧光检测细胞骨架蛋白和黑素小体分布。结果:p21siRNA转染黑素细胞后明显抑制p21 mRNA和蛋白表达(P<0.05)。与阴性siRNA+H2O2组比较,p21 siRNA+H2O2组β-半乳糖苷酶染色阳性率降低、细胞增殖能力增加及p21蛋白表达降低(P<0.05)。与对照组比较,阴性siRNA+H2O2组树突负性调节因子Rho A蛋白表达升高,而正性调节因子Rac1及Cdc42蛋白表达均降低,且细胞骨架蛋白及黑素小体荧光强度均降低(P<0.05)。经p21siRNA预处理后,p21 siRNA+H2O2组较阴性siRNA+H2O2组Rho A蛋白表达降低,Rac1和Cdc42蛋白表达均增高,且细胞骨架蛋白及黑素小体荧光强度均增高(P<0.05)。结论:低浓度H2O2诱导提前衰老的黑素细胞存在黑素传递功能障碍,p21 siRNA可改善低浓度H2O2诱导的黑素细胞提前衰老以及受损的黑素传递功能。

Objective:To investigate the effects of p21 siRNA through inhibiting p21 expression on premature senescence induced by a low concentration hydrogen peroxide(H2O2),as well as the relationship between pre-senescence and melanosomes transfer in human melanocytes.Methods:Melanocytes were transfected with p21 siRNA,and the inhibitory effects were measured by fluorescence microscope,qRT-PCR and western blot.Normal human melanocytes were collected and divided into 4 groups as follows:control group(without p21 siRNA transfection),p21 siRNA group(with p21 siRNA transfection),negative siRNA+H2O2 group(without p21 siRNA transfection,with 400μmol/L H2O2 after 24 hours)and p21 siRNA+H2O2 group(with p21 siRNA transfection and with 400μmol/L H2O2,24 hours after transfection).The effects of p21 siRNA treatment on pre-senescent melanocytes were assessed by SA-β-gal staining,EdU cell proliferation test and western blot.The expressions of dendritic regulatory proteins RhoA,Rac1 and Cdc42 were detected by western blot.The mRNA expression of p21 was detected by qRT-PCR.Cell cytoskeletal and the distribution of melanosomes were examined by immunofluorescence and fluorescence microscope.Results:The m RNA and protein expression of p21 was significantly inhibited after p21 siRNA transfection(P<0.05).Compared with negative siRNA+H2O2 group,the percentage of SA-β-gal staining was reduced,the cell proliferation rate was increased,and the p21 protein expression was reduced in p21 siRNA+H2O2 group(P<0.05).Compared with control group,the level of RhoA protein expression was increased,the RhoA and Cdc42 protein expression were reduced,the fluorescence intensity of F-actin was decreased in negative siRNA+H2O2 group(P<0.05).Compared with negative siRNA+H2O2 group,the RhoA protein expression was decreased,the Rac1 and Cdc42 protein expression was increased,and the fluorescence intensity of F-actin,NKI-beteb and melanosome were increased in p21 siR NA+H2O2 group(P<0.05).Conclusion:Melanocytes with premature senescence induced by a low concentration H2O2 show impaired melanosome transfer.The p21 siRNA can improve premature senescence induced by a low concentration H2O2 and associated impaired melanosome transfer in melanocytes.