| Objective:Lymphedema is a chronic progressive disease characterized by local swelling and functional impairment.However,there is no appropriate treatment for lymphedema and the existing conservative treatment and surgical treatment are uncertain.Here we aimed to investigate the role and mechanisms of human umbilical cord stem cell-derived exosomes?hucMSC-Ex?in lymphangiogenesis and lymphedema,which will provide a more effective and safe method for the treatment of lymphedema.Methods:1.hucMSC-Ex were extracted from the culture medium of hucMSCs via exosome extraction kit.Then the structure of hucMSC-Ex were identified by transmission electron microscope,Nanosight nanoparticle tracer analyzer.The surface markers CD9 and CD63 were detected by Western blot.Mice tail lymphedema model was established by resection of lymphatic vessels in the tail.hucMSC-Ex were injected into the site of tail lymphedema tisse,and hucMSC-Ex localization in injured tail was observed by in vivo imaging system.2.Tail diameters of the injured area of the mice tail and 1cm?D+10?and 2cm?D+20?from the incision edge were measured at the 7,14,21,28,35 and 42days before and after modeling respectively.3.The repair of edema and the changes in the number of lymphatic vessels were observed by HE staining and immunohistochemical staining of tissue sections.4.HucMSCs were overexpressed with Ang-2,and their culture supernatants were collected to extract hucMSC-ExAng-2ng-2 via exosome extraction kit.The same operation was conducted to extract hucMSC-Exsh-Ang-2.5.The ability of proliferation,migration and tube formation of HDLECss were observed after 48 hours of co-culturing with hucMSC-Ex?hucMSC-ExAng-2ng-2 or hucMSC-Exsh-Ang-2.6.HDLECs were overexpressed or knocked down with Ang-2 to observe the changes in their proliferation ability,migration ability and tube formation ability.7.Western blot and cellular immunofluorescence staining were used to detect the expression of Ang-2,LYVE-1,Prox1,and VEGFR3/p-Akt in HDLECs treated with hucMSC-Ex?hucMSC-ExAng-2ng-2 or hucMSC-Exsh-Ang-2.Results:hucMSC-ex could effectively repair lymphedema in the mouse tail,and the measurement of the diameter of four points in the tail showed significant postoperative edema at injured area,D+20,and D+30,and the edema was significantly reduced after hucMSC-Ex treatment.HE staining showed that hucMSC-Ex group revealed significant subcutaneous edema regression,decreased lymphatic swelling,and significantly thinner skin thickness in the tail.Immunohistochemical staining showed that the number of LYVE-1?+?lymphatic vessels was significantly increased after repair.After co-cultured with hucMSC-Ex or hucMSC-ExAng-2ng-2 for 48h,the abilities of proliferation,migration and tube formation of HDLECs were enhanced,in which hucMSC-ExAng-2ng-2 group was stronger than hucMSC-Ex group,and hucMSC-Ex group was stronger than normal group.Moreover,after co-cultured with hucMSC-Exsh-ang-2,the proliferation,migration and tube-forming abilities of hucMSC-Exsh-ang-2h-ang-2 group were significantly weaker than that of hucMSC-Ex group.After the overexpression of Ang-2 in HDLECs,proliferation,migration and lymphangiogenesis were enhanced.After the knocking down of Ang-2,the abilities of proliferation,migration and lymphangiogenesis were weakened.Western blot showed that hucMSC-Ex contained a large amount of Ang-2 protein,and the Ang-2,LYVE-1,Prox1 and VEGFR3 levels of HDLECs were significantly up-regulated after hucMSC-Ex treatment,and the fluorescence results were consistent with this phenomenon.In the study of mechanism,Western blot showed that p-Akt level was significantly up-regulated,while the total Akt level was not significantly changed.Conclusion:hucMSC-Ex may play a role in repairing lymphedema by promoting the expression of Prox1 and activation of Akt/VEGFR3 pathway by transporting Ang-2. |
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