中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (31): 4937-4944.doi: 10.12307/2024.704

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

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

李瑞博1，2，孔  宁1，2，孙  蕾3，马保东3，靳冉冉3，张文进2，岳  寒4，张  辉2   

  1. 1新乡医学院，河南省新乡市   453003；郑州大学附属郑州中心医院，2神经外科，3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
  • 收稿日期:2023-08-15 接受日期:2023-09-25 出版日期:2024-11-08 发布日期:2024-01-22
  • 通讯作者: 张辉，硕士，主任医师，郑州大学附属郑州中心医院神经外科，河南省郑州市 450007 岳寒，博士，主任医师，河南省人民医院干细胞再生医学中心，河南省郑州市 463599
  • 作者简介:李瑞博，男，1997年生，河南省许昌市人，汉族，新乡医学院在读硕士，医师，主要从事慢性意识障碍以及干细胞相关研究。
  • 基金资助:
    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

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

Li Ruibo1, 2, Kong Ning1, 2, Sun Lei3, Ma Baodong3, Jin Ranran3, Zhang Wenjin2, Yue Han4, Zhang Hui2   

  1. 1Xinxiang Medical University, Xinxiang 453003, Henan Province, China; 2Department of Neurosurgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Henan Province, China; 3Stem Cell Regenerative Medicine Translational Center, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Henan Province, China; 4Stem Cell Regenerative Medicine Center, Henan Provincial People’s Hospital, Zhengzhou 463599, Henan Province, China
  • Received:2023-08-15 Accepted:2023-09-25 Online:2024-11-08 Published:2024-01-22
  • Contact: Zhang Hui, Master, Chief physician, Department of Neurosurgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Henan Province, China Yue Han, MD, Chief physician, Stem Cell Regenerative Medicine Center, Henan Provincial People’s Hospital, Zhengzhou 463599, Henan Province, China
  • About author:Li Ruibo, Master candidate, Physician, Xinxiang Medical University, Xinxiang 453003, Henan Province, China; Department of Neurosurgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Henan Province, China
  • Supported by:
    Henan Medical Science and Technology Research Program Joint Construction Project, No. LHGJ20191045 (to ZH); Key Research & Development and Promotion Project of Henan Province, No. 222102310032 (to YH); Henan Medical Science and Technology Research Program Joint Construction Project, No. LHGJ20220858 (to ZWJ)

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摘要：


文题释义：

表观遗传修饰：通过化学修饰改变染色体上的DNA和蛋白质，从而对基因表达进行调控，影响基因的转录、翻译、核小体组装和染色质结构等多个层面，进而影响细胞的生理、病理过程。
染色体靶向切割和标签化(cleavage under targets and tagmentation，CUT&Tag)：一种用来研究蛋白质和DNA之间的相互作用，并确定目标蛋白质DNA结合位点的方法，是Chip-seq的衍生技术。


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成功构建稳定敲低REST的SH-SY5Y细胞，REST mRNA 转录水平和蛋白表达均降低；⑦缺血缺氧处理敲低REST的SH-SY5Y细胞与EPO-MSCs共培养后细胞凋亡显著增加、H3K36me3表达显著降低，转录组测序显示炎性相关基因Aldh1l2、Cth等以及凋亡抑制基因Mapk8ip1、Sod2在mRNA 转录水平表达降低(P < 0.01)；⑧结果表明：EPO-MSCs通过分泌组的改变影响H3K4me2的峰度从而激活REST、TET3的表达，上调H3K36me3的修饰水平，进而调控炎性相关基因Aldh1l2、Cth等以及凋亡抑制基因Mapk8ip1、Sod2的表达，促进神经元的存活。

https://orcid.org/0009-0000-8262-8966 (李瑞博) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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

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.

Key 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

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