摘要
目的:探究羟基红花黄色素A(HSYA)对人脐带间充质干细胞(hUC-MSC)复制性衰老的作用及机制。方法:通过连续体外扩增构建hUC-MSC复制性衰老模型,以P2代细胞为对照组,P10代细胞为衰老组。将衰老的细胞置于含有HSYA的培养基中培养,利用CCK-8和Incucyte S3动态活细胞成像分析系统分别检测细胞活力和细胞融合度,选取适宜浓度和时间进行后续实验。选取0.01 mg/ml的HSYA预处理衰老的细胞,衰老相关β-半乳糖苷酶(SA-β-gal)染色评估细胞衰老状况,qPCR法检测端粒相对长度,荧光探针DCFH-DA染色检测细胞内活性氧水平,JC-1染色法检测线粒体膜电位的变化,实时荧光定量qPCR法检测p53、p16、p21、OCT4和SOX2基因的表达,Western blot检测p53、p16、OCT4和SOX2蛋白的表达。结果:HSYA可以显著降低衰老细胞的SA-β-gal染色阳性率,抑制端粒的损耗,减少衰老细胞中活性氧的积累,提高线粒体膜电位,下调p53和p16基因的表达,上调OCT4基因的表达。HSYA也显著降低了p16蛋白的表达水平,提高了OCT4和SOX2蛋白的表达水平。结论:HSYA可能通过调控p53和p16信号通路,抑制氧化应激反应,改善hUC-MSC的复制性衰老。
Objective:To investigate the effects and mechanisms of hydroxysafflor yellow A(HSYA)on replicative senescence in human umbilical cord mesenchymal stem cells(hUC-MSCs).Methods:hUC-MSCs were cultured to construct a replicative senescence model through continuous amplification in vitro.Cells at passage 2 served as the control group,while cells at passage 10 were designated as the senescence group.The senescent cells were cultured in a culture medium containing HSYA.Cell viability was detected by the CCK-8 assay,and cell confluence was analyzed using the Incucyte S3 live-cell analysis system.The optimal concentration and time point were determined and utilized for subsequent experiments.Senescent cells were pretreated with 0.01 mg/ml HSYA,and the proportion of senescence-associatedβ-galactosidase(SA-β-gal)positive cells was detected to assess the senescence state.The relative telomere length was detected by qPCR.Reactive oxygen species(ROS)levels were measured using the fluorescent probe DCFH-DA.Mitochondrial membrane potential was assessed by JC-1 staining.The expression of p53,p16,p21,OCT4,and SOX2 genes was detected by qPCR.The expression of p16,p53,OCT4,and SOX2 proteins was analyzed by Western blot.Results:HSYA significantly decreased the SA-β-gal positive staining rate,inhibited telomere attrition,reduced the ROS accumulation,increased mitochondrial membrane potential in senescent cells.Additionally,HSYA downregulated the expression of p53 and p16,and upregulated the expression of OCT4.HSYA decreased p16 protein level and increased OCT4 and SOX2 protein levels.Conclusion:HSYA may ameliorate replicative senescence in hUC-MSCs by modulating the p53 and p16 signaling pathways and suppressing oxidative stress.
作者
汪思云
朱琦
谭春霞
鲁放
卢涛
WANG Si-Yun;ZHU Qi;TAN Chun-Xia;LU Fang;LU Tao(School of Life Science,Beijing University of Chinese Medicine,Beijing 102488,China)
出处
《中国实验血液学杂志》
北大核心
2025年第5期1507-1515,共9页
Journal of Experimental Hematology
基金
国家自然科学基金面上项目(52173276)。
关键词
羟基红花黄色素A
人脐带间充质干细胞
衰老
氧化应激
hydroxysafflor yellow A
human umbilical cord mesenchymal stem cell
senescence
oxidative stress
作者简介
共同第一作者:汪思云;共同第一作者:朱琦;通信作者:卢涛,教授.E-mail:taolu@bucm.edu.cn。
