中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (36): 7752-7761.doi: 10.12307/2025.750

• 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人脐带间充质干细胞对缺血缺氧SH-SY5Y细胞凋亡的影响

孔  宁1，2，唐吉祥1，2，侯豫博1，2，孟  澜2，3，孙  蕾4，马保东4，邵一鸣4，靳冉冉4，岳  寒5，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，4干细胞再生医学转化中心，河南省郑州市  450007；3郑州大学，河南省郑州市  450001；5河南省人民医院干细胞再生医学中心，河南省郑州市  450004
  • 收稿日期:2024-08-09 接受日期:2024-09-21 出版日期:2025-12-28 发布日期:2025-03-06
  • 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； 共同通讯作者：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:孔宁，男，1997年生，河南省周口市人，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项目(242102310112)，项目负责人：邵一鸣；河南省医学科技攻关项目(242102310144)，项目负责人：张辉

Effects of human umbilical cord mesenchymal stem cells overexpressing erythropoietin on apoptosis of SH-SY5Y neurons in ischemia and hypoxia

Kong Ning1, 2, Tang Jixiang1, 2, Hou Yubo1, 2, Meng Lan2, 3, Sun Lei4, Ma Baodong4, Shao Yiming4, Jin Ranran4, Yue Han5, Zhang Hui2   

  1. 1Xinxiang Medical College, Xinxiang 453003, Henan Province, China; 2Department of Neurosurgery, 4Stem Cell Regenerative Medicine Translation Center, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Henan Province, China; 3Zhengzhou University, Zhengzhou 450001, Henan Province, China; 5Stem Cell Regenerative Medicine Center, Henan Provincial People’s Hospital, Zhengzhou 450004, Henan Province, China
  • Received:2024-08-09 Accepted:2024-09-21 Online:2025-12-28 Published:2025-03-06
  • Contact: Zhang Hui, MS, Chief physician, Department of Neurosurgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Henan Province, China; Co-corresponding author: Yue Han, MD, Chief physician, Stem Cell Regenerative Medicine Center, Henan Provincial People’s Hospital, Zhengzhou 450004, Henan Province, China
  • About author:Kong Ning, Master candidate, Physician, Xinxiang Medical College, Xinxiang 453003, Henan Province, China; Department of Neurosurgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Henan Province, China
  • Supported by:
    Henan Provincial Medical Science and Technology Research Project, No. 242102310112 (to SYM); Henan Provincial Medical Science and Technology Research Project, No. 242102310144 (to ZH)

PDF

331

摘要：

文题释义：

人脐带间充质干细胞：是一种多能性干细胞，具备分化成多种不同细胞类型的潜力。人脐带间充质干细胞在神经系统疾病具有广泛的临床应用前景。
长链非编码RNA：是指长度超过200个核苷酸且不具备蛋白质编码功能的RNA转录本。长链非编码RNA XIST与神经系统疾病关系密切，近年来研究表明它可能参与调控缺血缺氧性脑病的病理生理过程，可以作为缺血缺氧脑病的潜在诊断和治疗靶点。

摘要
背景：长链非编码RNA(LncRNA)在神经系统发育及神经性疾病中扮演着重要角色，课题组前期研究证明了缺血缺氧环境下过表达促红细胞生成素的人脐带间充质干细胞(EPO-MSCs)具有更好的神经保护功能，且显著激活了LncRNA XIST的表达。研究表明XIST与缺血缺氧脑病的发病机制有关，但其受EPO-MSCs调控进而保护缺血缺氧神经元的作用和机制尚不清楚。
目的：探讨LncRNA XIST响应EPO-MSCs信号对缺血缺氧SH-SY5Y细胞凋亡影响的新机制。
方法：①通过慢病毒转染构建敲低LncRNA XIST(sh-XIST)和阴性对照(NC-XIST)SH-SY5Y细胞株，采用氧-葡萄糖剥夺诱导细胞缺氧缺血损伤，使用Transwell小室构建EPO-MSCs和sh-XIST、NC-XIST缺血缺氧SH-SY5Y细胞非接触共培养体系，使用CCK-8方法检测SH-SY5Y细胞的增殖能力，使用细胞划痕实验检测SH-SY5Y细胞的迁移能力，使用流式细胞仪检测SH-SY5Y细胞凋亡情况。②RNA-seq生信分析筛选sh-XIST与NC-XIST细胞株差异表达基因和通路，利用双荧光素酶实验验证miR-124-3p与靶基因XIST和GRIN1之间的关系，通过qRT-PCR验证下游miR-124-3p、GRIN1基因的表达水平。③加入miR-124-3p抑制剂和模拟物验证缺血缺氧并与EPO-MSCs共培养后SH-SY5Y的表型变化。
结果与结论：①与NC-XIST组相比，sh-XIST组SH-SY5Y细胞缺血缺氧损伤并与EPO-MSCs共培养后的增殖和迁移能力下降，细胞凋亡增加。②双荧光素酶实验结果显示，miR-124-3p与其靶基因XIST之间存在相互作用。在缺血缺氧条件下，转染miR-124-3p mimics的SH-SY5Y细胞在与EPO-MSCs共培养后表现出细胞凋亡减少的情况；相反，当SH-SY5Y细胞转染miR-124-3p inhibitor时，在相同的缺血缺氧条件及与EPO-MSCs共培养的情况下，细胞凋亡则显著增加。③通过对sh-XIST 进行转录组测序和生物信息学分析筛选得到显著下调的神经活性配体-受体通路及中枢神经系统发育关键受体基因GRIN1。④双荧光素酶实验结果显示，miR-124-3p与GRIN1相互作用，与NC-XIST组相比，缺血缺氧后sh-XIST组SH-SY5Y细胞GRIN1表达显著下调。结果表明，LncRNA XIST通过上调miR-124-3p从而促进GRIN1表达，进而减少缺血缺氧并与EPO-MSCs共培养SH-SY5Y细胞凋亡，增强其增殖、迁移能力；sh-XIST可以阻断这一保护功能。

关键词: hypoxic-ischemic encephalopathy, erythropoietin, umbilical cord mesenchymal stem cell, long non-coding RNA, gene modification, XIST, miR-124-3p, GRIN1, engineered stem cells

Abstract: BACKGROUND: Long non-coding RNA (LncRNA) plays an important role in nervous system development and neurological diseases. Previous studies by the research team have demonstrated that human umbilical cord mesenchymal stem cells overexpressing erythropoietin (EPO-MSCs) under ischemic and hypoxic conditions have better neuroprotective functions and significantly activate the expression of LncRNA XIST. Research suggests that XIST is related to the pathogenesis of hypoxic-ischemic encephalopathy, but the role and mechanism of its regulation by EPO-MSCs in protecting ischemic-hypoxic neurons remain unclear.
OBJECTIVE: To explore the new mechanism by which LncRNA XIST, in response to EPO-MSC signaling, affects the apoptosis of ischemic-hypoxic SH-SY5Y cells.
METHODS: (1) SH-SY5Y cell lines with knockdown of LncRNA XIST (sh-XIST) and negative control (NC-XIST) were constructed through lentiviral transfection. Oxygen-glucose deprivation was used to induce ischemic-hypoxic injury in the cells. Transwell chambers were used to create a non-contact co-culture system with EPO-MSCs, sh-XIST, and NC-XIST ischemic-hypoxic SH-SY5Y cells. Cell proliferation ability was detected using the CCK-8 assay. Cell migration ability was assessed using the scratch assay, and cell apoptosis was measured by flow cytometry. (2) RNA-seq bioinformatics analysis was performed to screen for differentially expressed genes and pathways between sh-XIST and NC-XIST cell lines. Dual-luciferase experiments were used to verify the relationship between miR-124-3p and the target genes XIST and GRIN1. qRT-PCR was conducted to validate the expression levels of downstream miR-124-3p and GRIN1 genes. (3) miR-124-3p inhibitors and mimics were added to verify phenotypic changes in SH-SY5Y cells after ischemic-hypoxic injury and co-culture with EPO-MSCs.
RESULTS AND CONCLUSION: (1) Compared with the NC-XIST group, SH-SY5Y cells in the sh-XIST group showed reduced proliferation and migration abilities and increased apoptosis after ischemic-hypoxic injury and co-culture with EPO-MSCs. (2) Dual-luciferase experiments showed that miR-124-3p interacted with the target gene XIST. SH-SY5Y cells transfected with miR-124-3p mimics and co-cultured with EPO-MSCs showed decreased apoptosis after ischemic-hypoxic injury, while SH-SY5Y cells transfected with miR-124-3p inhibitors showed increased apoptosis after co-culture with EPO-MSCs. (3) Transcriptomic sequencing and bioinformatics analysis of sh-XIST revealed significant downregulation of the neuroactive ligand-receptor pathway and the key receptor gene GRIN1 for central nervous system development. (4) Dual-luciferase experiments showed that miR-124-3p interacted with GRIN1. GRIN1 expression was significantly downregulated in the sh-XIST group after ischemic-hypoxic injury compared with the NC-XIST group. These findings indicate that LncRNA XIST promotes GRIN1 expression by upregulating miR-124-3p, thereby reducing cell apoptosis after ischemic-hypoxic injury and co-culture with EPO-MSCs and enhancing proliferation and migration. sh-XIST can block this protective function.

Key words: 缺血缺氧脑病, 促红细胞生成素, 脐带间充质干细胞, 长链非编码RNA, 基因修饰, XIST, miR-124-3p, GRIN1, 工程化干细胞

中图分类号: