过表达促红细胞生成素脐带间充质干细胞抑制缺血缺氧SH-SY5Y细胞凋亡及机制  

Erythropoietin-overexpressed umbilical cord mesenchymal stem cells inhibit neuroapoptosis in ischemic-hypoxic SH-SY5Y and its mechanism

作　　者：李瑞博 孔宁 孙蕾 马保东 靳冉冉 张文进 岳寒 张辉 Li Ruibo;Kong Ning;Sun Lei;Ma Baodong;Jin Ranran;Zhang Wenjin;Yue Han;Zhang Hui(Xinxiang Medical University,Xinxiang 453003,Henan Province,China;Department of Neurosurgery,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450007,Henan Province,China;Stem Cell Regenerative Medicine Translational Center,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450007,Henan Province,China;Stem Cell Regenerative Medicine Center,Henan Provincial People’s Hospital,Zhengzhou 463599,Henan Province,China)

机构地区：[1]新乡医学院,河南省新乡市453003 [2]郑州大学附属郑州中心医院神经外科,河南省郑州市450007 [3]郑州大学附属郑州中心医院干细胞再生医学转化中心,河南省郑州市450007 [4]河南省人民医院干细胞再生医学中心,河南省郑州市463599

出　　处：《中国组织工程研究》2024年第31期4937-4944,共8页Chinese Journal of Tissue Engineering Research

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20191045),项目负责人:张辉;河南省重点研发与推广专项(222102310032),项目负责人:岳寒;河南省医学科技攻关计划联合共建项目(LHGJ20220858),项目负责人:张文进。

摘　　要：背景:前期研究成功构建过表达促红细胞生成素的脐带间充质干细胞(erythropoietin-overexpressed umbilical cord mesenchymal stem cells,EPO-MSCs),发现其可以显著减少缺血缺氧人神经母细胞瘤细胞株(SH-SY5Y)细胞的凋亡。目的:探究EPO-MSCs对缺血缺氧SH-SY5Y细胞可能的神经保护机制及其相关表观遗传学机制。方法:用氧-葡萄糖剥夺法缺血缺氧诱导SH-SY5Y细胞损伤,分别与慢病毒转染空载质粒的脐带间充质干细胞(NC-MSCs)、EPO-MSCs共培养后进行转录组测序,根据组间差异基因进行相关分析。同时应用多因子法检测缺血缺氧性脑病患者和对照组脑脊液上清及共培养细胞上清炎性因子表达水平。蛋白组学检测NC-MSCs、EPO-MSCs差异表达蛋白。应用染色体靶向切割和标签化(CUT&Tag)测序技术检测基因组H3K4me2修饰情况,并与转录组测序联合分析。慢病毒载体感染构建稳定敲低REST的SH-SY5Y细胞,应用qRT-PCR、Western blot检测REST的表达水平,然后再缺血缺氧处理并与EPO-MSCs共培养,流式细胞仪检测细胞凋亡情况、Western blot检测H3K36me3组蛋白的表达差异,然后进行转录组测序分析差异表达基因。结果与结论:①与对照组比较,缺血缺氧性脑病患者脑脊液上清中单核细胞趋化蛋白1、白细胞介素6、白细胞介素18、白细胞介素1β、干扰素α2、白细胞介素23水平显著增加(P<0.01);②缺血缺氧SH-SY5Y细胞与EPO-MSCs共培养后单核细胞趋化蛋白1、白细胞介素6表达水平明显降低;③蛋白质网络相互作用分析发现单核细胞趋化蛋白1、白细胞介素6相关调控蛋白CXCL1、BGN等显著下调;④转录组测序分析发现EPO-MSCs组SH-SY5Y细胞中促炎基因较NC-MSCs组显著下调,组蛋白修饰的功能富集以及转录因子REST、TET3的表达显著上调;⑤转录组测序与CUT&Tag联合分析发现表观遗传水平变化以及转录因子REST和TET3启动子区域显著激活;⑥成功构建稳定�BACKGROUND:Previous studies have successfully constructed erythropoietin-overexpressed umbilical cord mesenchymal stem cells.It was found that the apoptosis of ischemic and hypoxic human neuroblastoma cell line(SH-SY5Y)was significantly reduced by erythropoietin-overexpressed umbilical cord mesenchymal stem cells.OBJECTIVE:To explore the possible neuroprotective mechanisms of erythropoietin-overexpressed umbilical cord mesenchymal stem cells against ischemichypoxic SH-SY5Y and their associated epigenetic mechanisms.METHODS:Oxygen-glucose deprivation was applied to ischemia-hypoxia-induced SH-SY5Y cell injury,and multifactorial assays were applied to detect the expression levels of inflammatory factors in the cells before and after hypoxia and co-culture,respectively,with mesenchymal stem cells,as well as lentiviraltransfected null-loaded plasmids of the negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression levels of supernatant inflammatory factors were detected by multifactor assay after co-culture.Proteomics was used to detect the differentially expressed proteins of negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.Cleavage under targets and tagmentation sequencing was applied to detect genomic H3K4me2 modification,and joint analysis was conducted with RNA-sequencing.Lentiviral vector infection was applied to construct the stable knockdown of REST in SH-SY5Y cells.qRT-PCR and western blot assay were performed to detect the expression level of REST.The apoptosis was detected by flow cytometry after co-culture of oxygen-glucose deprivation treatment with erythropoietinoverexpressed umbilical cord mesenchymal stem cells.The expression difference of H3K36me3 group proteins was detected by western blot assay,and transcriptome sequencing was performed to analyze the differentially expressed genes.RESULTS AND CONCLUSION:(1)Compared with the control group,monocyte chemotactic protein 1,i

关 键 词：脐带间充质干细胞 组蛋白修饰 缺血缺氧性脑病 基因修饰 染色体靶向切割和标签化 神经元限制性沉默因子 单核细胞趋化蛋白1 白细胞介素6 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学] R542.2