[1]班跃耀,孙蕾,马保东,等.促红细胞生成素基因修饰的人脐带间充质干细胞对神经元周期和凋亡的调控作用及机制[J].新乡医学院学报,2023,40(10):909-916.[doi:10.7683/xxyxyxb.2023.10.002]
　BAN Yueyao,SUN Lei,MA Baodong,et al.Regulatory effect and mechanism of human umbilical cord mesenchymal stem cells modified with erythropoietin gene on cell cycle and apoptosis of neuron[J].Journal of Xinxiang Medical University,2023,40(10):909-916.[doi:10.7683/xxyxyxb.2023.10.002]
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促红细胞生成素基因修饰的人脐带间充质干细胞对神经元周期和凋亡的调控作用及机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
40卷
期数:
2023年10
页码:
909-916
栏目:
基础研究
出版日期:
2023-10-05

文章信息/Info

Title:
Regulatory effect and mechanism of human umbilical cord mesenchymal stem cells modified with erythropoietin gene on cell cycle and apoptosis of neuron
作者:
班跃耀1; 2; 孙蕾3; 马保东2; 靳冉冉2; 李瑞博1; 2; 孔宁1; 2; 张辉2
（1.新乡医学院研究生院，河南　新乡　453003；2.郑州市中心医院神经外科，河南　郑州　450007；3.郑州大学，河南　郑州　450007）
Author(s):
BAN Yueyao1; 2; SUN Lei3; MA Baodong2; JIN Ranran2; LI Ruibo1; 2; KONG Ning1; 2; ZHANG Hui2
（1.Graduate School,Xinxiang Medical University,Xinxiang 453003,Henan Province,China；2.Department of Neurosurgery,Zhengzhou Central Hospital,Zhengzhou 450007,Henan Province,China;3.Zhengzhou University,Zhengzhou 450007,Henan Province,China）
关键词:
间充质干细胞; 促红细胞生成素; 神经元; 缺氧缺血性脑病; 神经保护
Keywords:
mesenchymal stem cells; erythropoietin; neuron; hypoxic-ischemic encephalopathy; neuroprotection
分类号:
R743.9
DOI:
10.7683/xxyxyxb.2023.10.002
文献标志码:
A
摘要:
目的　探讨促红细胞生成素（EPO）基因修饰的人脐带间充质干细胞（hUC-MSCs）对神经元周期和凋亡的调控作用及机制。
方法　将对数生长期SH-SY5Y细胞随机分为MSCs组、正常对照（NC）-间充质干细胞（MSCs）组及EPO-MSCs组，MSCs组细胞不做任何转染，NC-MSCs组及EPO-MSCs组细胞分别转染对照病毒空载体和过表达EPO慢病毒载体。应用荧光显微镜和流式细胞术验证NC-MSCs组及EPO-MSCs组细胞转染效率，应用Western blot检测MSCs组、NC-MSCs组及EPO-MSCs组细胞中EPO蛋白表达，实时荧光定量聚合酶链反应检测MSCs组、NC-MSCs组及EPO-MSCs组细胞中EPO mRNA表达，酶联免疫吸附法检测MSCs组、NC-MSCs组及EPO-MSCs组细胞上清液中 EPO 蛋白表达。将SH-SY5Y细胞分为常氧组和缺氧缺血组，常氧组细胞正常培养，缺氧缺血组细胞置于含体积分数0.5%O2三气体培养箱中24 h制备缺氧缺血细胞模型；将缺氧缺血SH-SY5Y细胞随机分为MSCs共培养组、NC-MSCs共培养组、EPO-MSCs共培养组，MSCs共培养组、NC-MSCs共培养组、EPO-MSCs共培养组缺氧缺血SH-SY5Y细胞分别与MSCs、NC-MSCs、EPO-MSCs共培养；应用流式细胞术检测各组细胞周期及凋亡情况。对MSCs共培养组、NC-MSCs共培养组、EPO-MSCs共培养组缺氧缺血SH-SY5Y细胞行RNA-Seq分析，并行富集京都基因和基因组百科全书(KEGG)通路富集。
结果　EPO-MSCs慢病毒转染效率为98.51%，NC-MSCs慢病毒转染效率为97.83%。EPO-MSCs组细胞中EPO蛋白和mRNA相对表达量显著高于MSCs组和NC-MSCs组（P<0.05）； MSCs组与NC-MSCs组细胞中EPO蛋白和mRNA相对表达量比较差异无统计学意义（P>0.05）。EPO-MSCs组细胞上清液中EPO蛋白表达水平显著高于MSCs组和NC-MSCs组（P<0.05）；MSCs组与NC-MSCs组细胞上清液中EPO表达水平比较差异无统计学意义（P>0.05）。缺氧缺血组SH-SY5Y细胞凋亡率及G0-G1期细胞比例显著高于常氧组（P<0.05）。EPO-MSCs共培养组SH-SY5Y细胞凋亡率及G0-G1期细胞比例显著低于MSCs共培养组和NC-MSCs共培养组（P<0.05）；MSCs共培养组与NC-MSCs共培养组SH-SY5Y细胞凋亡率及G0-G1期细胞比例比较差异无统计学意义（P>0.05）；MSCs共培养组、NC-MSCs共培养组和EPO-MSCs共培养组SH-SY5Y细胞凋亡率及G0-G1期细胞比例显著低于缺氧缺血组（P<0.05）。 RNA-Seq分析显示，促分裂素原活化蛋白激酶（MAPK）信号通路和细胞凋亡通路是NC-MSCs共培养缺氧缺血SH-SY5Y细胞、EPO-MSCs共培养缺氧缺血SH-SY5Y细胞中富集程度最高的通路，p53途径的激活在细胞凋亡的调节中起关键作用。
结论　EPO基因修饰的MSCs可显著抑制缺氧缺血神经元凋亡，减少细胞间期阻滞，增强MSCs对缺氧缺血性脑病的保护作用，其机制可能与EPO基因修饰的MSCs激活MAPK-p53信号通路相关。
Abstract:
Objective　To investigate the regulatory effect and mechanism of erythropoietin (EPO) gene modified human umbilical cord mesenchymal stem cells (hUC-MSCs) on cell cycle and apoptosis of neuron.
Methods　The SH-SY5Y cell line in logarithmic growth phase was randomly divided into MSCs group,normal control(NC)- mesenchymal stem cells (MSCs) group and EPO-MSCs group.The cells in the MSCs group did not undergo any transfection,while the cells in the NC-MSCs group and EPO-MSCs group were transfected with the control virus empty vector and overexpressed EPO lentivirus vector,respectively.The transfection efficiency of cells in the NC-MSCs group and EPO-MSCs group were verified by using fluorescence microscopy and flow cytometry.The expression of EPO protein in the cells in the MSCs group,NC-MSCs group and EPO-MSCs group was detected by using Western blot;the expression of EPO mRNA in cells in the MSCs group,NC-MSCs group and EPO-MSCs group was detected by real time fluorescence quantitative polymerase chain reaction;the expression of EPO protein in cell culture medium in the MSCs group,NC-MSCs group and EPO-MSCs group was detected by enzyme linked immunosorbent assay.SH-SY5Y cells in logarithmic growth phase were divided into normoxic group and hypoxic-ischemic group.The SH-SY5Y cells in the normoxic group were cultured normally,while the SH-SY5Y cells in the hypoxic-ischemic group were exposed to 0.5% O2 for 24 hours to prepare hypoxic-ischemic SH-SY5Y cell models;the hypoxic-ischemic SH-SY5Y cells were randomly divided into MSCs co-culture group,NC-MSCs co-culture group and EPO-MSCs co-culture group;the hypoxic-ischemic SH-SY5Y cells in the MSCs co-culture group,NC-MSCs co-culture group and EPO-MSCs co-culture group were co-cultured with MSCs,NC-MSCs and EPO-MSCs,respectively;the cell cycle and apoptosis in each group were detected by flow cytometry.RNA-Seq analysis was performed on ischemic-hypoxic SH-SY5Y cells in the MSCs co-culture group,NC-MSCs co-culture group and EPO-MSCs co-culture group,and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed.
Results　The transfection efficiency of EPO-MSCs lentivirus was 98.51%,while the transfection efficiency of NC-MSCs lentivirus was 97.83%.The relative expression levels of EPO protein and mRNA in cells in the EPO-MSCs group were significantly higher than those in the MSCs group and NC-MSCs group (P<0.05);there was no statistically significant difference in the relative expression levels of EPO protein and mRNA between the MSCs group and the NC-MSCs group (P>0.05).The expression level of EPO protein in cell culture medium in the EPO-MSCs group was significantly higher than that in the MSCs group and NC-MSCs group (P<0.05);there was no statistically significant difference in the expression level of EPO protein in cell culture medium between the MSCs group and the NC-MSCs group (P>0.05).The apoptosis rate of SH-SY5Y cells and the proportion of G0-G1 phase cells in the hypoxic-ischemic group were significantly higher than those in the normoxic group (P<0.05).The apoptosis rate and proportion of G0-G1 phase cells of SH-SY5Y cells in the EPO-MSCs co-culture group were significantly lower than those in the MSCs co-culture group and NC-MSCs co-culture group (P<0.05);there was no statistically significant difference in the apoptosis rate and proportion of G0-G1 phase cells of SH-SY5Y cells between the MSCs co-culture group and the NC-MSCs co-culture group (P>0.05);the apoptosis rate and proportion of G0-G1 phase cells of SH-SY5Y cells in the MSCs co-culture group,NC-MSCs co-culture group and EPO-MSCs co-culture group were significantly lower than those in the hypoxic-ischemic group (P<0.05).RNA-Seq analysis showed that the mitogen-activated protein kinases (MAPK) signaling pathway and apoptosis pathway were the most enriched pathways in NC-MSCs co-cultured hypoxic-ischemic SH-SY5Y cells and EPO-MSCs co-cultured hypoxic-ischemic SH-SY5Y cells.The activation of the p53 pathway plays a key role in regulating cell apoptosis.
Conclusion　EPO gene modified MSCs can significantly inhibit apoptosis of hypoxic-ischemic neurons,reduce intercellular phase arrest,and enhance the protective effect of MSCs on hypoxic-ischemic encephalopathy.The mechanism may be related to the activation of MAPK-p53 signaling pathway by EPO gene modified MSCs.

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更新日期/Last Update: 2023-10-05