Study On The Effects Of Polymorphonuclear-derived Exosomes On Angiogenesis

Objective: The aim of the study was to explore the role of polymorphonuclear-derived exosome(PMN-Exo)in promoting the expression of angiogenesis-related factors of bone marrow stromal cell(BMSC)and human umbilical vein endothelial cell(HUVEC),and the subcutaneous ectopic angiogenesis in nude mice.Methods:Bone marrow of C57 mice was obtained by irrigating medullary cavity.PMN from bone marrow was extracted by immunomagnetometry,and determined by flow cytometry(FCM).In the same way,BMSC was extracted from human jaw bone through medullary cavity irrigation,and identified by FCM.PMN was cultured with BMSC and HUVEC for 20 h,then the cell migration ability was detected by scratch experiment,and the tube formation effect of HUVEC was observed after a 12-hour culture.After 24 h of co-culture,the expression levels of angiogenesis-related factors(VEGF,PDGF)were detected by quantitative polymerase chain reaction(qPCR)and western blot;The supernatants of PMN were collected after 24 h of culture.Then,the exosomes in the supernatants were extracted by overspeed centrifugation.Electron microscope(TEM)was used to observe its morphological structure.Nanoparticle tracking analysis(NTA)was used to detect its size and concentration.Membrane protein markers CD9,CD81,TSG-101 were also detected by western blot.The above three methods were all carried out to co-verify it.After labelled with Dil,PMN-Exo was co-cultured with BMSC and HUVEC to observe their endocytosis by confocal laser scanning microscopy(CLSM).PBS group and PMN-Exo group were included in the later experiments.After the addition of PMN-Exo to BMSC and HUVEC for 20 h,the cell migration ability was detected by scratch experiment,and the tube formation effect of HUVEC was observed after a 12-hour culture.After the addition for 24 h,the cell proliferation ability was detected by Ed U method.the expression levels of angiogenesis-related factors VEGF,PDGF were detected by qPCR and western blot after 5 d;Hydrogel and PMN-Exo complex were constructed at the low temperature,and the retention effect of PMN-Exo was further detected by NTA.The compound was aseptically injected into the subcutaneous surface of the back of nude mice,and the samples were collected after one week.The angiogenesis effect in vivo was observed by HE staining and CD31 staining.Results:PMN and BMSC were extracted and identified successfully.The results of the scratch experiment showed that the migration ability of BMSC and HUVEC in the PMN group increased significantly compared with non-PMN group.After adding PMN,the tube forming effect of HUVEC was significantly enhanced.The results of qPCR and western blot also showed that the angiogenesis-related factors VEGF,PDGF in the PMN group were significantly increased;Based on the above results,PMN-Exo was further extracted and successfully identified by morphology observation,particle size analysis and membrane protein detection,and the endocytosis effect of it was verified by Dil staining.The results of the scratch experiment showed that the proliferation and migration ability of BMSC and HUVEC in the PMN-Exo group increased significantly when compared with the PBS group.After the addition of PMN-Exo,the tube-forming effect of HUVEC was significantly enhanced,and angiogenesis-related factors VEGF and PDGF were also significantly increased;PMN-Exo has a retention effect in the gel from the result of NTA.The results of HE staining and CD31 staining also showed that the number of blood vessels and the angiogenesis effect in the PMNExo/hydrogel complex group increased significantly when compared with the PBS/hydrogel complex group.Conclusion:1.PMN can effectively enhance the migration ability of h BMSC and HUVEC,as well as the tubulogenesis effect of HUVEC,and promote the expression of angiogenesis-related factors of h BMSC and HUVEC.2.PMN-Exo can also effectively enhance the migration and proliferation ability of h BMSC and HUVEC,as well as the tubulogenesis effect of HUVEC,and promote the expression of angiogenesis-related factors of h BMSC and HUVEC.3.PMNExo/hydrogel complex can effectively improve the capacity of angiogenesis in vivo and promote subcutaneous ectopic angiogenesis in nude mice.