人脐带间充质干细胞来源的外泌体对神经细胞缺血缺氧损伤修复机制研究  

Repair mechanism of human umbilical cord mesenchymal stem cell-derived exosome on neuronal ischemia and hypoxia injury

作　　者：胡昌龙[1] 周文勤 葛璐[3] 樊永忠[1] 王鹏 Hu Changlong;Zhou Wenqin;Ge Lu;Fan Yongzhong;Wang Peng(Neurosurgery Department,People's Hospital of Danyang,Danyang 212300,China;Emergency Department,Zhenjiang First People's Hospital,Zhenjiang 210000,China;Gastroenterology Department,People's Hospital of Danyang,Danyang 212300,China)

机构地区：[1]江苏省丹阳市人民医院神经外科,212300 [2]江苏省镇江市第一人民医院急诊医学科,212000 [3]江苏省丹阳市人民医院消化科,212300

出　　处：《中华急诊医学杂志》2020年第7期934-940,共7页Chinese Journal of Emergency Medicine

基　　金：江苏省镇江市社会发展项目(SH2017033);镇江市社会发展指导性项目(FZ2017007,FZ2018033);丹阳市科技发展专项(SF201702)。

摘　　要：目的探讨人脐带间充质干细胞来源的外泌体(human umbilical cord mesenchymal stem cell-derived exosome,hucMSC-ex)对缺血缺氧神经细胞增殖、迁移、凋亡、自噬等的影响及其机制。方法培养原代神经胶质细胞,建立氧糖剥夺(oxygen-glucose deprivation,OGD)模型,hucMSC-ex孵育之后,MTT检测细胞增殖抑制率,流式细胞技术检测细胞凋亡,RT-PCR检测凋亡基因mRNA表达水平及Western blot检测其对凋亡蛋白、自噬蛋白及PI3K/AKT信号表达变化。计量资料多组间比较采用方差分析,组间两两比较采用SNK-q检验。结果MTT实验结果显示OGD可抑制细胞增殖,hucMSC-ex孵育2 h后,细胞增殖抑制率较对照组下降(P<0.05);流式细胞技术检测结果显示hucMSC-ex孵育之后可以减少细胞凋亡(P<0.05);细胞迁移实验显示OGD可降低细胞迁移能力(P<0.01),外泌体孵育之后细胞迁移能力增强(P<0.05)。RT-PCT技术及Western Blot检测结果显示,OGD可以诱导细胞发生凋亡与自噬,hucMSC-ex可激活PI3K/Akt信号通路,抑制Bax与Caspase-3蛋白(P<0.05)及mRNA(P<0.01)表达水平,促进Bcl-2蛋白(P<0.05)及mRNA(P<0.01)表达水平;同时,hucMSC-ex可抑制Beclin-1、Atg3和LC3-Ⅱ蛋白表达水平(P<0.05)及Beclin-1、Atg3基因mRNA表达水平(均P<0.01)。结论hucMSC-ex可促进OGD神经胶质细胞的增殖、迁移,抑制其凋亡及自噬水平,对缺血缺氧损伤性神经胶质细胞具有修复能力,其保护机制与PI3K/Akt信号通路相关。Objective To investigate the effects of human umbilical cord mesenchymal stem cell-derived exosome(hucMSC-ex)on proliferation,migration,apoptosis and autophagy in ischemia-anoxia neurons,and to provide a theoretical study for clinical research on stroke.Methods Primary glial cells were cultured and OGD model was established.Then,these cells were incubated with huMSC-exosome.The inhibition rate of proliferation was detected by MTT assay.Apoptosis was observed by flow cytometry.The expressions of apoptosis related proteins were confirmed by RT-PCR and Western blot.The expressions of autophagy related proteins and PI3K/Akt signal were observed by Western blot.The data were analyzed using SPSS 17.0 software,multiple-group comparisons were performed using one-way ANOVA,and SNK-q test was used for pairwise comparison between groups.Results MTT assay showed that OGD could inhibit cell proliferation of primary glial cells.After incubation with hucMSC-ex for 2 h,the inhibition rate of cell proliferation was lower than that of the control.The flow cytometry technology showed that hucMSC-ex reduced cell apoptosis.The cell migration experiments showed that OGD reduced cell migration capacity,but cell migration increased after exosomal incubation.RT-PCT and Western blot showed that OGD induced autophagy and apoptosis,hucMSC-ex activated PI3K/Akt signaling pathway,inhibited the expression of Bax and Caspase-3(both P<0.05),and promoted the expression of Bcl-2(P<0.05).hucMSC-ex inhibited the expression of Beclin-1,Atg3 and LC3-Ⅱ(al lP<0.01).Conclusions huMSC-exosome promote the proliferation and migration in ischemia-anoxia-injured neurons and inhibit the apoptosis and autophagy.The mechanism that hucMSC-ex repaired the injured nerve cells might be associated with PI3K/Akt signaling pathway.

关 键 词：神经缺血缺氧损伤 外泌体 凋亡 PI3K/ AKT信号通路 

分 类 号：R743.3[医药卫生—神经病学与精神病学]