| ObjectiveThe purpose of study is to investigate the effect of extracellular vesicles secreted by bone marrow mesenchymal stem cells(BMSC-EVs)on the proliferation and migration of RSC96 Schwann cells,as well as possible mechanisms.This research work will provide new therapeutic strategies for the diagnosis and treatment of peripheral nerve injury.Methods(1)Bone marrow mesenchymal stem cells were isolated purified identified.(2)Extracellular vesicles secreted by bone marrow mesenchymal stem cells were isolated and identified by ultracentrifugation method.(3)Red fluorescent dye(CM-Dil)-labeled BMSC-EVs were incubated with RSC96 cells for 4 hours,and the fusion process of EVs and RSC96 membranes was observed under fluorescence microscope.(4)The effect of BMSC-EVs on proliferation of RSC96 cells was detected by MTT assay and plate colony formation assay.(5)The effect of BMSC-EVs on the migration of RSC96 cells wasdetected by test and Trans-well assay.(6)Flow cytometry analysis was used to detect the effect of BMSC-EVs on apoptosis of RSC96 cells.(7)Western-blot was conducted to detect the expression of related proteins.(8)MTT assay,Transwell assay,and Western-blot detection were performed to measure the changes of proliferation,migration and related protein expression of RSC96 cells after ERK inhibitor U0126 treatment.Results(1)BMSCs were successfully isolated and cultured.The morphology of the cells was long spindle-shaped and whirlpool-like.The results of flow cytometry showed BMSCs with CD29,CD90,CD105 expression but without CD19.Osteogenic induction displayed a positive result,and adipogenic induction produced lipid droplets.(2)The size of BMSC-EVs ranged from 80 to 400 nm with an average of142 nm.A circular or elliptical membrane structure can be observed under a transmission electron microscope.(3)Fusion of BMSC-EVs with RSC96 cell membranes were confirmed by Dir-labeled assay.(4)MTT assay and plate colony formation assays showed thatBMSC-EVs inhibited RSC96 cell proliferation.(5)Scratch test and Transwell assay showed that BMSC-EVs inhibited migration of RSC96 cells.(6)The expression level of Bcl-2 was down-regulated after treatment with BMSC-EVs,and the level of Bax was up-regulated.(7)Flow cytometry analysis revealed that BMSC-EVs induced RSC96 cells apoptosis.(8)After treament of RSC96 cells with U0126 ERK activity was increased with,the down-regulation of Bcl-2 increased,the up-regulation of Bax decreased,and RSC96 cell proliferation and migration were inhibited after BMSC-EVs treatment.ConclusionsBMSC-EVs inhibit the proliferation and migration of RSC96 cells,finally induce apoptosis by activating the ERK pathway. |
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