过氧化氢诱导间充质干细胞衰老模型的建立

Establishment of an aging model of mesenchymal stem cells

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。

Objective To establish an aging model of mesenchymal stem cells （MSCs） and to investigate aging related biological mechanism for the purpose of studying the senesence of MSCs .Methods MSCs were separated and purified from human placenta, and the cells of the third passage（P3-MSCs） were cultured in the medium for 2 hours, then 100,200 and 300 μmol/L hydrogen peroxide （ H2 O2 ） was added to the cells for 2 hours to establish the MSCs aging model in vitro. Biological characteristics of aging MSCs were evaluated by cell cycle assay and senescence associated β-galactosidase staining.The expression of p16,p21 and p53 genes was further measured using quantitative real-time PCR （RT-PCR）.Re-sults Compared with the control , the number of MSCs treated with 200μmol/L H2 O2 for 2 hours was significantly decreased and the cells displayed less adipogenic ,osteogenic and chondrogenic differentiation .Moreover ,after exposure to 200 μmol/L H2 O2 , the majority of the cells were in the G 0/G1 phase as showed by cell cycle analysis .The percentage of senescence-associated β-galactosidase-positive cells was increased , and the expression of p 16 , p21 and p53 mRNA and protein was significantly increased.Conclusion The results of this study has demonstrated that the H 2 O2 （200 μmol/L） can be used to establish the aging model of MSCs in vitro, and the cellular phenotypic alteration may attribute to the cell cycle associated gene expression （p16, p21, and p53）.