摘要
背景:细胞移植治疗受区存在氧化应激过程,影响疗效。研究氧化应激对牙髓干细胞的影响及机制方面的报道较少。目的:分离培养人牙髓干细胞,分析加入过氧化氢(H_2O_2)对其衰老程度的影响。方法:牙髓干细胞分为3组,分别用PBS、100μmol/L H_2O_2、200μmol/L H_2O_2刺激2 h。倒置显微镜下观察细胞形态,β-半乳糖苷酶染色观察细胞衰老程度,BrdU试剂盒和细胞计数法检测细胞增殖能力,免疫荧光检测细胞骨架排列情况和sirt1蛋白的表达,Western Blot法测定sirt1、p16蛋白的表达。结果与结论:(1)牙髓干细胞呈成纤维细胞样梭形外观。(2)H_2O_2刺激后,细胞体积增大、β-半乳糖苷酶染色加深、细胞增殖能力降低,随着H_2O_2浓度增加,牙髓干细胞衰老增强,在此过程中,p16表达上调,sirt1表达受到抑制。(3)结果表明H_2O_2刺激促进牙髓干细胞衰老,sirt1、p16参与了此过程,为牙髓干细胞移植提供了理论基础和实验支持。
BACKGROUND： The process of oxidative stress that impacts the curative effect exists in the region which accepts cell transplantation. However, there are few reports about the effects of oxidative stress on human dental pulp stem cells and relevant mechanism. OBJECTIVE： To understand the effect of hydrogen peroxide on the senescence of human dental pulp stem cells. METHODS： Human dental pulp stem cells were isolated and cultured in PBS, 100 and 200 μmol/L hydrogen peroxide for 2 hours, respectively. Cell morphology was observed under inverted microscope, degree of cell senescence monitored by β-galactosidase staining, cell proliferation ability detected by Brd U kit and cell counting method, cytoskeleton of dental pulp stem cells and expression of sirt1 tested using immunofluorescence method, and expression of sirt1 and p16 proteins measured by western blot assay. RESULTS AND CONCLUSION： Dental pulp stem cells exhibited a fibroblast-like morphology with spindle-shaped appearance. After stimulated by hydrogen peroxide, the cell volume was enlarged, the β-galactosidase staining deepened and the proliferation of dental pulp stem cells reduced. The enhancement of senescence of dental pulp stem cells was accompanied with the increasing concentration of hydrogen peroxide, and in this process, the expression of p16 was raised while the expression of sirt1 was decreased. In conclusion, the senescence of human dental pulp stem cells can be promoted by the stimulation of hydrogen peroxide, and sirt1 and p16 are involved in this process. Our findings may provide a theoretical and experimental foundation for autologous transplantation of dental pulp stem cells.
出处
《中国组织工程研究》
CAS
北大核心
2016年第10期1481-1487,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金青年基金项目(81500809)~~
