An Observational Study Of Exosomes Derived From Bone Mesenchymal Stem Cell Promoting Angiogenesis Of Human Umbilical Vein Endothelial Cells Under Hypoxia

Objective:Over the years,cell-based therapeutic angiogenesis has been widely adopted to treat ischemic diseases,such as coronary artery diseases,cerebral infarction and critical limb ischemia.As a type of stem cells with multi-differentiation function,bone mesenchymal stem cells(BMSCs)are widely adopted in the treatment of acute myocardial infarction and in ischemic microenvironments,Some studies have shown that its efficacy is partly due to its paracrine action,but its mechanism is still to be investigated.The purpose of the present research is to extract and identify BMSCs-EXO,so as to observe their usefulness in the angiogenesis of human umbilical vein endothelial cells(HUVECs)under hypoxic condition.Methods:1.BMSCs were extracted from male SD rats(primary generation)aged 3-4weeks,and cultured in DMEM with 10% serum,1% double antibody and 1% b FGF solution.The growth curve was then observed through CCK8 method,and its osteogenic differentiation ability was observed after alizarin red and Von Kossa staining;2.Exosomes(BMSCs-EXO)were isolated from the upper culture medium of BMSCs by ultra-high speed centrifugation;3.After applying Western blots,the BMSCs-EXO was transilluminated through electron microscope and analyzed through laser particle size analyzer for identifica-tion.4.According to the experimental design,HUVEC was split into normoxic culture environment group(Control),hypoxic culture environment group(Hypoxia),and hypoxic + BMSCs-EXO group(Hypoxia + EXO).The Control group cells were cultured in a normoxic(21% O2,5% CO2)cell incubator at 37 ?,while cells in Hypoxia group were cultured in a cell incubator containing 3% O2 at 37 ?.For Hypoxia+EXO group cells,10ug/ml BMSCs-EXO was added to HUVECs before entering the hypoxic incubation box;5.Endothelial cell function test:The migration effect of BMSCs-EXO on HUVECs cells under hypoxic condition were observed through cell wound scratch assays and Trans Well tests;6.Angiogenesis test:The effect of BMSCs-EXO treatment on the angiogenesis of HUVECs cells under hypoxic condition were observed through angiogenesis experiment;7.Angiogenesis-related protein expression test:The influence of BMSCs-EXO treatment on the expression of vascular endothelial growth factor(VEGF)proteins in HUVECs were observed using Western Blot method.Results:1.The staining observation of HE in BMSCs showed that the cells were long fusiform-shaped and fibroblast-like.The cell count results showed that the growth curve of BMSCs increased at the beginning and then decreased,and reached the peak at the 7th day.Cell surface markers CD29 and CD34 were negative,while CD45 and CD90 were positive.After osteogenic induction and differentiation,both alizarin red staining and Von Kossa staining showed a large number of black mineralized nodules.They both attested that BMSCs had the potential to differentiate into osteocytes.2.The research group successfully extracted BMSCs-EXO and the transillumi-nation results of electron microscopes showed that BMSCs-EXO had a bilayer membrane structure.The Zetasizer nano zs90 instrument was adopted for analysis,and calculation results showed that the exosomal particle size was 65.2 ± 2.9nm(n=3).Western blot results showed that the expression of HSP70,CD63 and other marker proteins in BMSCs-EXO was positive.3.Endothelial cell function test showed:By analyzing the scratch area,the scratch healing speed of Hypoxia + EXO group was faster than that of Control group and Hypoxia group(P < 0.001).The results of Transwell experiment suggested that the number of HUVECs transmembrane cells in Hypoxia + EXO group was significantly higher than that in Hypoxia group(P < 0.01).It is suggested that BMSCs-EXO can promote the HUVECs migration under hypoxic condition.4.Angiogenesis test showed:The results of HUVECs tube formation experiment showed that the number of extracorporeal tubules generated in Hypoxia + EXO group was significantly higher than that in Control group and Hypoxia group(P < 0.05).5.Angiogenesis-related protein expression test showed:Western blot results showed that the expression of VEGF proteins in Hypoxia + EXO group was significantly higher than that in Control group and Hypoxia group(P < 0.05).Conclusions:This study found that BMSCs-EXO can promote angiogenesis of HUVECs under hypoxic condition,which may be connected with the up-regulated VEGF expression.