D-半乳糖诱导正常人口腔黏膜角质形成细胞衰老的研究

Study of D-galactose Induced Senescence of normal human oral keratinocytes

目的:明确D-半乳糖对正常人口腔黏膜细胞(normal human oral keratinocytes,NHOKs)促衰老作用,并探索潜在机制。方法:体外分离培养NHOKs,角形蛋白和波形蛋白免疫细胞化学染色法鉴定NHOKs。分别以10、20、40、80g/L D-半乳糖处理诱导细胞作为实验组,正常培养液组作为对照组。通过CCK-8法检测细胞增殖活力,采用衰老相关β半乳糖苷酶染色(senescence-associated-β-galactosidase,SA-β-gal)分析细胞衰老情况,Western blot检测衰老相关蛋白p53和p21表达水平。活性氧(reactive oxygen species,ROS)检测试剂盒检测细胞内氧化应激水平,Western blot检测氧化应激相关蛋白sirt1的表达。结果:D-半乳糖抑制NHOK细胞增殖,具有时间和浓度依赖性。D-半乳糖增加SA-β-gal染色阳性率,上调衰老相关基因p53和p21,显著升高细胞内ROS,降低氧化应激相关蛋白sirt1表达。结论:D-半乳糖可诱导NHOKs发生衰老,该过程可能与细胞氧化应激有关。

Objective:To investigate the senescence effect of D-galactose(D-gal)on normal human oral keratinocytes(NHOKs)and explore the potential mechanism.Methods:NHOKs were isolated and cultured in vitro and identification was detected by immunocytochemistry.NHOKs were treated with different concentrations of D-galactose(10,20,40,80 g/L)for12 h,24 h,48 h,as experimental group,while control group was cultured by normal medium.Cell proliferation activity was detected by CCK-8 method.Senescence-associated-β-galactosidase(SA-β-gal)staining and Western blot of senescence related proteins p53 and p21 were detected for analysis of cell senescence.ROS detection kit was used to detecthe intracellular oxidative stress level,and Western blot was used to detect the expression of the oxidative stress-related protein sirt1.Results:D-galactose reduced NHOKs proliferation in a time-and-dose dependent manner.Compared with control group,the quantity of SA-β-gal positive cell increased,as well as the expression of p53 and p21.ROS production in NHOKs treated by D-gal was significantly increased.Meanwhile the expression of sirt1 was decreased.Conclusion:D-galactose can induce senescence of NHOKs,which may be related to oxidative stress of cells.