过氧化氢诱导间充质干细胞衰老模型的建立

发布时间：2018-07-08 08:43

本文选题：胎盘 + 间充质干细胞　；参考：《军事医学》2015年05期

【摘要】：目的建立间充质干细胞(MSCs)衰老模型,探讨其衰老的相关生物学改变,从而为衰老相关研究提供有效的细胞模型工具。方法取新生胎盘组织利用酶消化法分离纯化获得人胎盘源间充质干细胞(MSCs)。将第3代细胞接种到培养皿。在完全培养基基础上分别加入终浓度100、200、300μmol/L的过氧化氢(H2O2)培养2 h,处理结束后更换为完全培养基继续培养24 h。进行MTT、细胞周期分析、分化诱导实验和β-半乳糖苷酶染色验证衰老MSCs模型的建立,应用RT-PCR法检测衰老相关基因p16、p21、p53 mRNA的表达变化。结果与对照组比较,200μmol/L H2O2作用2 h,倒置显微镜下见MSCs形态较为宽大扁平;MTT光密度值显著下降,流式检测结果显示细胞周期处于G0/G1期细胞比例增加;成脂、成骨和成软骨分化能力减弱;β-半乳糖苷酶阳性细胞百分比显著升高;p16、p21、p53 mRNA表达水平显著升高。结论 200μmol/L H2O2作用2 h可建立MSCs体外衰老模型,该模型细胞的生物学变化可能是由p16、p21、p53细胞周期蛋白的活性调节引起的。
[Abstract]:Objective to establish an aging model of mesenchymal stem cells (MSCs) and to explore the biological changes related to its senescence, so as to provide an effective tool for the study of aging. Methods Human placental mesenchymal stem cells (MSCs) were isolated and purified by enzyme digestion. The third passage cells were inoculated into a petri dish. Hydrogen peroxide (H _ 2O _ 2) with final concentration of 100200300 渭 mol / L was added to the complete culture medium for 2 h, and then replaced with the complete medium for 24 h. MTT, cell cycle analysis, differentiation induction test and 尾 -galactosidase staining were used to verify the establishment of aging MSCs model. RT-PCR was used to detect the expression of p16p21 p21 p53 mRNA. Results compared with the control group, 200 渭 mol / L H _ 2O _ 2 treatment for 2 h resulted in a significant decrease in MTT optical density of MSCs under inverted microscope. Flow cytometry showed that the proportion of cells in G _ 0 / G _ 1 phase was increased. The percentage of 尾 -galactosidase positive cells increased significantly, and the expression of p16 p21 and p53 mRNA increased significantly. Conclusion 200 渭 mol / L H _ 2O _ 2 for 2 h can induce the senescence model of MSCs in vitro, and the biological changes of the model cells may be caused by the regulation of cyclin activity in p16 p21 ~ (-1) p53 cells.
【作者单位】：北京工业大学生命科学与生物工程学院;军事医学科学院放射与辐射医学研究所;
【分类号】：R329.2

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