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

Objective To study the impact of exosomes derived from human umbilical mesenchymal stem cells on cardiac function and myocardial fibrosis in dilated cardiomyopathy Rats.Methods1.Building and identificating of DCM rats100 rats which have the same weight male SD rats were seperated into normal group(n=20)and DCM group(n=80)randomly.DCM rats are injected Adriamycin(1mg/kg)by tail vein two times per week for 8 weeks to build DCM model rats.After stopping 8-weeks treatment,we use the echocardiography to detect the cardiac function.2.Culture of hUCMSCs,isolation exosome from hUCMSCs(hUCMSCs-Ex)and identificationUsing tissue block method to culture hUMSCs primary and flow cytometry(FCM)to detect the surface markers of P4 generation.Collecting exosomes-free medium of hUMSCs to seperate exosomes.Detecting the exosomes by transmission electron microscopy,Western blot and Nanoparticle Tracking Analysis(NTA).3.The effects of injection of exosomes derived from human umbilical cord mesenchymal stem cells via tail vein on the myocardial fibrosis and myocardial function in rats with Adriamycin-induced DCMThe successful established DCM model rats were divided into 4 groups randomly:the low-dose group of hUCMSCs-Ex(n=17),medial-dose group of hUCMSCs-Ex(n=17),the high-dose group of hUCMSCs-Ex(n=17)and the DCM control group(n=17),which were received hUCMSCs-Ex 20μg/kg,100μg/kg,250μg/kg and 1 ml/kgPBS,respectively,and followed by once time a week for 28 days.After 28-days injection,the rats were killed,the left ventricular tissue were left for pathological detection by immunohistochemistry by immunohistochemistry including HE and Masson staining.The level of myocardial fibrosis was detected by RT-PCR and Western blot.Results1.Establishment and identification of the rat model of DCMDCM group of rats have different heart failure symptom such as decreased activity,losing of appetite,ascites and moulting.Echocardiography showed that dilated ventricular cavity and incongruity of the ventricular wall activity.The heart dimensions(LVEDD and LVESD)were significantly increased(p < 0.01),while LVEF and LVFS were significantly decreased in the DCM group compared to the normal group(p < 0.01).2.hUCMSCs and hUCMSCs-Ex isolating and identificatingThe cut up umbilical cord tissue were inoculated into culture bottle.After about 15 days the primary hUCMSCs group up from the side of the tissue like polygonal or spindly shapes.FCM detect found the protein expression of the CD90 was positive,but that of CD146 and HLA-DR was negative.Transmission electron micrographs found that hUCMSCs-Ex are small oval vesicles with low electron densities.Western blot found the positive expression of CD81 and CD9 protein.3.Effects of hUCMSCs-Ex on cardiac function and myocardial fibrosisAfter treatment of hUCMSCs-Ex,heart failure symptom in the dose groups of rats reduced,and the survival rate increased.Before injection of hUCMSCs-Ex,compared to the normal group,LVEF and LVFS significantly reduced in the DCM control group(P<0.01).After treatment of hUCMSCs-Ex,LVEF and LVFS is higher in doses of hUCMSCs-Ex groups compared with the DCM control group(P <0.05).Compared with the DCM control group,the level of mRNASmad2、mRNACOLⅠ、m RNAα-SMA is lower after the treatment of hUCMSCs-Ex.Western blot analysis showed that the lower expression of Smad2、COLⅠ、α-SMA in myocardium after the treatment of hUCMSCs-Ex;Masson staining showed more blue-stained collagen deposition among the tissue in DCM control group compared with normal group.However the level of fibrosis has improved significantly after the treatment of hUCMSCs-Ex;HE staining of DCM controlgroup showed that myocardial cells and interstitial edema,myocardial cells disorganized arrangement,and inflammatory cell increase;the situation has improved significantly after the treatment of hUCMSCs-Ex.Conclusion1.The DCM rat model could be established by adriamycin via tail vein injection with several times and small doses successfully.The clinical manifestation and pathological mechanism of successful DCM rats is same with clinical DCM.2.Treatment of hUCMSCs-Ex via tail vein can reduce the process of myocardial fibrosis in DCM rats and increase cardiac function.The paracrine mechanism of stem cells was further verification.