Effects Of Exosomes Derived From Human Umbilical Cord-derived Mesenchymal Stem Cells On Cardiac Function And Microvascular Regeneration In A Rat Model Of Adriamycin-induced Dilated Cardiomyopathy

Objective To explore the effect of exosomes secreted by human umbilical mesenchymal stem cells on microvascular regeneration in dilated cardiomyopathy Rats.Methods1.Establishment and identification of the rat model of DCMTotally 140 male SD rats were randomly divided into control group(n=20)and DCM group(n=120).Rats in the DCM groups receiving Adriamycin(1 mg/kg)intraperitoneally twice a week for 8 weeks to establish DCM models.One week after stopping injection,echocardiography was performed to evaluate the cardiac function.While myocardial tissue underwent HE and Masson staining.2.Isolation and culture of hUCMSCs and exosome from hUCMSCs purification and identificationThe hUMSCs were primary cultured by tissue block method and the surface markers of P3 generation was detected by using flow cytometry(FCM).The serum-free medium of hUMSCs was collected to extract exosomes that was ngidentified the morphology by transmission electron microscopy.Surface specific maker protein CD81 and CD9 were detected via Western blot.3.The effects of intramuscularly grafted human umbilical cord mesenchymal stem cells on the myocardial collagen in rats with adriamycin-induced DCMRats in the DCM groups receiving adriamycin(1 mg/kg)intraperitoneally twice a week for 8 weeks to establish DCM models.The DCM rats were randomly divided into 4groups: the low-dose group of hUCMSCs-exo(n=16),the high-dose group ofhUCMSCs-exo(n=16)and medial-dose group of hUCMSCs-exo(n=16),the blank control group,which were received,20 ? g/kg hUCMSCs-exo,100 ? g/kg hUCMSCs-exo,250?g/kg hUCMSCs-exo and 1 ml/kg PBS,respectively,and followed by once time a week for four weeks.After 4 weeks,all groups of rats were put to death.After four weeks,the rats were killed,the left ventricular myocytes were taken for pathological section,and the microvessel density of myocardium was detected by immunohistochemistry and their myocardial tissue underwent HE and Masson staining.Results1.Establishment and identification of the rat model of DCMDCM group of rats were found different level of heart failure performance such as depressed,reduced activity and food intake,ascites formation,claw and tail edema.Echocardiography showed that expanded heart chamber,diffuse weakening and incongruity of the wall activity.As compared to the control group,the heart dimensions(LVEDd and LVEDD)(p < 0.01 for both time points)were significantly increased,while LVEF and LVFS were significantly decreased in the DCM group(p < 0.01 for both time points).Myocardial tissue HE staining of DCM group showed that myocardial cells and interstitial edema,myocardial fibers arranged in disorder,fracture or necrosis partly,and focal lymphocytes infiltration among myocardial fibers;Masson staining showed a large number of green-stained collagen deposition among the myocardial fibers,not only around blood vessels.2.hUCMSCs and exosome from hUCMSCs purification and identificationThe hUCMSCs cultured from the minced umbilical cord tissue were inoculated into culture in Petri dishes.The hUCMSCs appeared as fibroblast-like cells attached to the sides of the Petri dishes with polygonal or spindly shapes.Staining of the hUCMSCs was positive for CD90 and CD73,but negative for CD34 and CD45.Transmission electron micrographs showed that hUCMSCs-ex are bi-lipid membrane vesicles with diameters ranging in size from 30 nm to 1 ?m with low electron densities.Western blot analysis confirmed that the surfaces of the hUCMSCs-ex were positive for the exosome-specific marker proteins CD81 and CD9.3.Effects of exosomes derived from human umbilical cord-derived mesenchymal stem cells on cardiac function and microvascular regenerationAfter injection of hUCMSCs-exo,heart failure performance in the high-dose and low-dose groups of rats improved,and the survival rate increased.Before administration of hUCMSCs-exo,the left ventricular ejection fraction(LVEF)and the left ventricular fraction shortening(LVFS)were significantly decreased in the DCM group compared to those of the control group(P <0.01).After injection via tail vein of hUCMSCs-exo,LVEF and LVFS of the various dose groups had significant difference compared with the DCM group(P <0.05),but no significant difference among the various dose groups(P >0.1).Compared with the model group,the normal group and the low dose group,the myocardial neovascularization density in the middle and high dose group were significantly different(p<0.01).Strong positive expression of CD34 for the micrangium in the myocardial tissue of middle-dose group and high-dose group rats were observed.Western blot analysis showed that effective expression of VEGF-2 in myocardium of rats with high dose was detected.myocardial microvascular endothelial injury is reduced significantly;Masson staining showed that the excretion of body dose group rat myocardial green staining of collagen deposition decreased;HE staining showed that the dose group compared with the DCM control group decreased inflammatory cell infiltration,cell edema,myocardial fibers were arranged in order.Conclusion1.The rat model of DCM could be successfully built by adriamycin intraperitoneal injection with fractional dose,which was highly consistent with laboratory characteristics and pathological results of clinical DCM.2.Injection of hUCMSCs-exo through tail vein can regulate the process of myocardial microvessel proliferation in DCM rats,protect myocardial cell function and improve cardiac function.The paracrine mechanism of stem cells was confirmed,but there was no significant difference between different doses of hUCMSCs-exo.