| BackgroudAdriamycin(ADM)is a classic drug in the combined chemotherapy of hematological malignancies.However myocardial toxicity has became a bottleneck in its clinical application.DZR and antioxidants are used to reduce the cardiac damage,but the effect is limit.Therefore,it is significant to find another way to reverse myocardial damage by ADM.Mesenchymal stem cells(MSCs)can be transformed into cardiomyocytes,and become the new therapy for heart diseases.Study shows that MSCs has therapeutic effects on myocardial damage by ADM.But The application of MSCs has limitations,such as long training time,large difference in efficiency of expansion in vitro,and need to be returned many times,and so on.So we need to study how to apply MSCs efficiently.Studies also show that MSCs are regulated by positive feedback of miR-21,which is the exocrine factor of MSCs and can enhance MSCs proliferation and differentiation.Based on relevant literatures and previous work of our groups,we predict that miR-21 overexpressing BMSCs can more effectively reverse the cardiac damage caused by ADM.Objective:Our study aims to compare the protective effects of bone marrow mesenchymal stem cells(BMSCs)before and after miR-21 transfection on adriamycin-induced cardiac damage in rats,and explore its possible mechanisms.Methods:1 To establish an animal model of adriamycin-induced myocardial damage:To determine the optimal dosage by studying the damage of different doses of ADM in rats.24 SD rats were randomly divided into 4 groups:control group,2mg group,3mg group and 4mg group.Six rats for each group.All the drugs were injected through the tail vein,q48h for 7 times.Groups were divided as follow:(1)Control group:were injected with the same amount of normal saline;(2)2mg group:were given adriamycin with 2 mg/kg;(3)3mg group:were given adriamycin with 3 mg/kg;(4)4mg group:were given adriamycin with 4 mg/kg;Then the general situation,survival,weight,cardiac coefficient,and myocardial pathology of the rats were observed.2 To compare the protective effects of bone marrow mesenchymal stem cells(BMSCs)on adriamycin-induced cardiac damage in rats before and after miR-21transfection:(1)Isolation and characterization of rat BMSCs;(2)PLVX-miR-21 virus was transfected into rat BMSCs.(3)In vitro trial.1)The BMSCs were divided into three groups:BMSCs group,empty plasmid group(transfected with pLVX)and miR-21 over-expression group(transfected with pLVX-miR-21-BMSCs).2)Characterization of pLVX-miR-21-BMSCs.3)To compare the miR-21 expression between BMSCs group,empty plasmid group and miR-21 over-expression group.4)To campare the migration of BMSCs by transwell assay before and after miR-21 transfection.5)To campare the proliferation of BMSCs by CCK-8 assays before and after miR-21 transfection.(4)Animal trial.50 SD rats were randomly divided into 5 groups:negative control group,ADM group,BMSCs group,NC group and miR-21 over-expression group.Ten rats for each group.All the drugs were injected through the tail vein.ADM with 2mg/kg q48h for 7times.BMSCs 1×10~^6 once on the fifteenth day.Groups were divided as follow:1)Negative control group(control group):were given the same amount of normal saline.2)ADM group:were given ADM only.3)BMSCs group:were given ADM and BMSCs.4)NC group:were given ADM and pLVX-BMSCs.5)miR-21 over-expression group:were given ADM and pLVX-miR-21-BMSCs.All of the rats were fed for 4 weeks at the same time.We observed the general situation of rats,then studied on myocardial damage,apoptosis and vascular regeneration:cardiac function evaluated by Doppler ultrasonography,myocardial pathology,detection of Cx-43,troponint,BNP by WB and Bcl-2,Bax,VEGF by PCR and VIII factor by immunohistochemical.Results(1)Compared with the control group,the growth rate of 2mg group was lower(P<0.05),the cardiac coefficient was higher(P<0.05),the myocardial fibers were fine,disordered,structure unclear,nucleus degeneration,cytoplasm vacuoles;The weight and the cardiac coefficient of 3mg group were decline(P<0.05),the myocardial fibers were trophic,nucleus necrosis,partial fused into pieces.All of the rats in 4mg group given the sixth dose of ADM died.(2)The characterization of BMSCs was not modified and the miR-21 expression was higher after miR-21 transfection.These confirmed that miR-21 had successfully infected the BMSCs.(3)MiR-21 overexpression enhanced the ability of migration of rat BMSCs by Transwell assay.(4)MiR-21 overexpression enhanced the ability of multiplication of rat BMSCs by CCK-8 assay.(5)The result of animal trial:(1)BMSCs improved the state of rats given ADM,especially in miR-21over-expression group;(2)Before transfection,compared with the control group,the values of FS,EF,LVEDD and LVESD of adriamycin groups were all significantly decreased(P<0.05).After transfection,compared with ADM group,the heart function of other groups improved.(3)Myocardial pathology also showed that the dissolution and scarring of the cells were reduced after the treatment of MSCs,especially in miR-21over-expression group.(4)Analysis of Bax,troponinT,BNP:Compared with the control group,adriamycin groups were higher(P<0.05);compared with ADM group,MSCs therapy groups were decreased(P<0.05);compared with BMSCs group,p miR-21 over-expression group was declined(P<0.05).(5)Analysis of Bcl-2,Cx-43,VEGF,VIII factor level:Compared with the control group,ADM groups were decreased(P<0.05);compared with ADM group,MSCs therapy groups were increased(P<0.05);compared with BMSCs group,miR-21 over-expression group was rised(P<0.05).Conclusion(1)The animal model of myocardial damage induced by ADM establishes sussfully.2mg/kg of ADM is the optimal dose.(2)The pLVX-miR-21 has successfully infected the rat BMSCs and doesn't affect the characterization of BMSCs.MiR-21 can enhance the ability of the miR-21expression,migration and multiplication of rat BMSCs in vitro.(3)BMSCs can improve the general situation,repair of myocardial injury and the cardiac function in rats which were given ADM,especially for the miR-21over-expression group.(4)The mechanism of miR-21 protection of myocardial is related to promote angiogenesis and anti-apoptosis. |
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