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.
作者
胡昌龙
周文勤
葛璐
樊永忠
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)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
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)。
