Effect Of Inhibiting The Release Of Exosomes On Proliferation Ability And Hypoxic Stress Tolerance Of Bone Marrow Mesenchymal Stem Cells

【Background】Myocardial infarction（MI）is widely considered to be one of the leading causes of mortality for patients with cardiovascular disease.However,current therapies for MI cannot guarantee satisfactory results.For all these years,scientists have been searching various treatments to promote cardiac repair and regeneration after MI.Of those,stem cell transplantation has gain ascendancy.At present,piles of research work had been devoted to explore mechanisms underlying the cardio-protective effects of bone marrow mesenchymal stem cells（BM-MSCs）and MSC-derived exosomes,while whether exosomes secretion would affect the biological characteristics of BM-MSCs has been of little concern.In this study,we investigated the effect of inhibiting the release of exosomes on the proliferation ability and hypoxic stress tolerance of BM-MSCs.【Objective】To investigate the effect of inhibiting the release of exosomes on proliferation ability and hypoxic stress tolerance of BM-MSCs and explore the underlying mechanisms.【Methods】1.Exosomes secretion deficient mouse model was constructed by knocking out gene Rab27a with TALEN technology.Then F1 mice were identified by phenotype observation,genotyping PCR,Sanger sequence and detection of Rab27a protein expression.2.To verify the effect of Rab27a deficiency on BM-MSCs released exosomes, BM-MSCs were isolated from both wild-type mice and Rab27a knockout（Rab27a KO）mice and cultured.BM-MSCs at passage 3-8 were used for subsequent experiments.The relative amount of MSCs was counted by crystal violet staining.Exosomes were extracted from the culture medium using total exosomes isolation kit and quantified by Nanoparticle Tracking Analysis（NTA）.The size and morphology of exosomes were observed by transmission electron microscopy（TEM）,and the exosomes special marker protein CD63 was detected by western blotting.3.To investigate the effect of defective secretion of exosomes from BM-MSCs on cell proliferation,the proliferation ability of BM-MSCs was evaluated by5-ethynyl-2’-deoxyuridine（Edu）assay and protein expression level of PCNA.4.To investigate whether inhibiting the secretion of BM-MSCs exosomes would affect the ability of hypoxia tolerance,BM-MSCs were treated with H₂O₂ at different concentrations（0,25,50,100,200,400μmmol/L）.TUNEL staining and MTS assay were used to detect the ability of MSCs to withstand hypoxia【Results】1.Rab27a KO mice presented with grayish-white hairs because of melanosome release deficiency at the root of the hair follicle.Genotyping PCR and Sanger sequencing showed that there was a 7-base deletion at the exon of Rab27a gene in Rab27a KO mice.And the protein expression of Rab27a was also significantly reduced in Rab27a KO mice.2.The relative amount of MSCs was counted by crystal violet staining,and exosomes were collected from cultured medium with total exosomes isolation kit.The relative amounts of exosomes secreted from a single MSC were quantified by NTA.Compared with WT-MSCs,KO-MSCs released exosomes were significantly reduced.3.Edu staining showed that the proliferation ability of KO-MSCs were significantly decreased compared with WT-MSCs.And the protein expression of proliferating cell nuclear antigen（PCNA）in KO-MSCs was significantly down-regulated.4.MTS assay was used to detect the cell viability of BM-MSCs treated with H₂O₂ at different concentrations.We found that exosome-release deficiency would lower the resistance of BM-MSCs to hypoxia stress tolerance.Compared with WT-MSCs,TUNEL staining showed that the apoptotic rates of KO-MSCs were significantly increased after exposure to H₂O₂.【Conclusion】Inhibition of exosomes release from the BM-MSCs results in significantly decreased proliferation ability and hypoxic stress tolerance...