中国组织工程研究2024，Vol.28Issue(31)：4937-4944,8.DOI:10.12307/2024.704
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
摘要
Abstract
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 ischemic-hypoxic 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 lentiviral-transfected 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 erythropoietin-overexpressed 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,interleukin-6,interleukin-18,and interleukin-1 beta,interferon α2,and interleukin-23 levels significantly increased in the cerebrospinal fluid supernatant of patients with ischemic-hypoxic encephalopathy(P<0.01).(2)After co-culturing SH-SY5Y cells with erythropoietin-overexpressed umbilical cord mesenchymal stem cells under ischemia and hypoxia,the expression levels of monocyte chemotactic protein 1 and interleukin-6 were significantly reduced.(3)Analysis of protein network interactions revealed significant downregulation of monocyte chemotactic protein 1,interleukin-6 related regulatory proteins CXCL1 and BGN.(4)Transcriptome sequencing analysis found that pro-inflammatory genes were down-regulated,and functional enrichment of histone modifications,and the expression of transcription factors REST and TET3 significantly up-regulated in the erythropoietin-overexpressed umbilical cord mesenchymal stem cell group compared with the negative control mesenchymal stem cell group.(5)Combined analysis of transcriptome sequencing and cleavage under targets and tagmentation revealed changes in epigenetic levels as well as significant activation of the promoter regions of transcription factors REST and TET3.(6)Stable knockdown REST in SH-SY5Y cells was successfully constructed;the transcript levels of REST mRNA and protein expression were both decreased.(7)After the REST knockdown SH-SY5Y cells were co-cultured with erythropoietin-overexpressed umbilical cord mesenchymal stem cells,apoptosis was significantly increased and H3K36me3 expression was significantly decreased.Transcriptome sequencing results showed that the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2 was reduced at mRNA transcription level(P<0.01).(8)It is concluded that erythropoietin-overexpressed umbilical cord mesenchymal stem cells activated the expression of REST and TET3 by altering the kurtosis of H3K4me2 and upregulated the modification level of H3K36me3,which in turn regulated the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2,and facilitated neuronal survival.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6Key words
umbilical cord mesenchymal stem cell/histone modification/ischemic-hypoxic encephalopathy/gene modification/chromosome targeted cleavage and labeling/neuron-restrictive silencer factor/monocyte chemotactic protein-1/interleukin-6分类
医药卫生引用本文复制引用
李瑞博,孔宁,孙蕾,马保东,靳冉冉,张文进,岳寒,张辉..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[J].中国组织工程研究,2024,28(31):4937-4944,8.基金项目
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 （LHGJ20191045）
河南省重点研发与推广专项(222102310032),项目负责人:岳寒 （222102310032）
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Henan Medical Science and Technology Research Program Joint Construction Project,No.LHGJ20191045(to ZH) （LHGJ20220858）
Key Research&Development and Promotion Project of Henan Province,No.222102310032(to YH) （to YH）
Henan Medical Science and Technology Research Program Joint Construction Project,No.LHGJ20220858(to ZWJ) （to ZWJ）
