Experimental Study On The Pro-angiogenesis Effect Of Extracellar Vesicles Derived From Adipose-drerived Stem Cells Pretreated With Hypoxia After Skin Flap Transplantation

Objectives: In recent years,human adipose-derived mesenchymal stem cells(hASCs)have been widely used in the field of tissue repair and reconstruction.A large number of studies have shown that the efficacy of hASCs is mainly due to the extracellular vesicles(EVs)produced by paracrine vesicles.Further research found that the function of EVs is affected by the microenvironment of hASCs.Therefore,this study simulated physiological hypoxic environment to cultivate hASCs in order to obtain hypoxic hASC-EVs.Then evaluated whether hypoxic hASC-EVs could improve the healing of flap transplantation and explored its mechanism.Methods: The experimental program is mainly divided into two parts:(1)In vitro,co-cultivation of normoxic hASC-EVs or hypoxic hASC-EVs(EVs released by hypoxia-pretreated hASC-EVs)with human umbilical vein endothelial cells(HUVECs).Using CCK-8,scratch test,Matrigel Matrigel tube formation test to evaluate their proliferation,migration and tube-like differentiation ability;Using SOD,MDA kit and flow cytometry to detect its improvement of oxidative stress and apoptosis ability.(2)In vivo,Randomly divided SD rats into 3 groups and made a 9 ×3 cm random flap on the back of the rat.Injected normal oxygen hASC-EVs,hypoxic hASC-EVs and PBS into the entire skin around the flap immediately.On the 7th postoperative day,the flap healing and angiogenesis were evaluated by gross observation,HE and CD31 immunohistochemistry.HIF-1? and VEGF histochemical results and their mRNA levels further revealed the mechanism of hypoxic hASC-EVs to promote flap healing.Results: In vitro,the results showed that hypoxic hASC-EVs have stronger proliferation,migration and tube-like differentiation ability than normal oxygen hASC-EVs.But the effect of improving cell oxidative stress and apoptosis is not obvious.In vivo,we found that hypoxic hASC-EVs had the best effect on promoting angiogenesis after flap transplantation.Hypoxia preconditioning can promote angiogenesis by up-regulating HIF-1? expression and promoting VEGF secretion.Conclusions: In summary,the results of the study showed that hypoxic hASC-EVs can upregulate the expression of HIF-1?,increase the secretion of VEGF,and then promote the formation of new blood vessels,improve the blood supply of flaps,increase the survival rate of flaps after flap transplantation.