SRT1720抗老年大鼠骨髓间充质干细胞衰老的作用及机制研究

SRT1720 ameliorates senescence of mesenchymal stem cells from aged rats

目的探究SRT1720抗老年大鼠骨髓间充质干细胞(MSCs)衰老的作用及其机制并建立一种可行的预处理方案。方法以CCK-8法检测细胞生存率,通过过氧化应激模型,确定SRT1720预处理方案。之后采用SRT1720预处理和溶剂DMSO预处理,以β-半乳糖苷酶染色法显示细胞衰老情况,荧光定量PCR法检测衰老相关的p16、p21 mRNA表达水平,Western blot法检测衰老相关的p16、p21蛋白表达水平。管腔形成试验分析预处理的细胞上清液重悬人脐静脉内皮细胞(HUVEC)管腔形成情况。采用活性氧检测试剂盒检测MSCs内活性氧类物质含量。免疫荧光法检测MSCs细胞内DNA损伤情况。结果最适宜的SRT1720预处理方案为1μM SRT1720预处理老年MSC 24h,在此条件下,细胞在氧化应激条件下生存率最高,48h后衰老相关的β-半乳糖苷酶染色阳性率明显下降,细胞衰老相关的p16、p21 mRNA和蛋白表达水平明显降低,细胞上清液促HUVEC管腔形成能力增强,细胞内活性氧类物质含量减少,DNA损伤反应减弱。结论 SRT1720预处理可通过减轻细胞内活性氧类物质的产生及DNA损伤而改善老年MSC衰老表型及旁分泌功能。

Objective To investigate the effect of SRT1720 on mesenchymal stem cells(MSCs)from aged rats and its mechanism.Methods The MSCs were obtained from SD rats of 18 months of age.Cell survival was detected by CCK-8 kit.The pretreatment scheme was decided by a peroxide stress model of aged MSCs.Aged MSCs were distributed into SRT1720 or DMSO pretreatment groups.The senescence associatedβ-gal staining was used to detect the senescence of aged MSCs.The expression of senescence associated genes was detected by quantitative RT-PCR,and the senescence associated proteins were quantified by Western blot.The tube formation test was used to estimate the pro-angiogenesis of the supernatant of pretreated aged MSCs.The reactive oxygen species(ROS)were detected by a ROS assay kit.The DNA damage of aged MSCs was detected by immunofluorescence method,respectively.Results The optimized pretreatment condition for aged MSCs was determined as 1μM SRT1720 for 24h by monitoring the survival of aged MSCs subjected to peroxide stress medium.Pretreatment with these conditions ameliorated the positive rate ofβ-gal staining of aged MSCs,and reduced the expression of senescence associated genes and proteins(p16/p21).The cell supernatant of aged MSCs pretreated with SRT1720 promoted the tube formation of human umbilical vein endothelial cells.ROS and DNA damage in aged MSCs were mitigated by SRT1720 pretreatment.Conclusion SRT1720 pretreatment could ameliorate the senescence and paracrine function of aged MSCs by reducing ROS and DNA damage.