Effect Of Transplantation Of SMHs Carrying MSCs Infected With Lentiviral Vectors Bearing MiR-499on The Treatment Of Acute Myocardial Infarction In Rats

Objective1. Investigate the role of miR-499in inducing the differentiation of MSCs intocardiomyocyte-like cells;2. Observe the effect of transplantation of SMHs carrying MSCs infected withlentiviral vectors bearing miR-499on cardiac function in rats with AMI and thesurvival, retention, differentiation of engrafted MSCs, etc.Methods1. After selected with the puromycin, miR-499lentivirus vector with GFPfluorescence and puromycin resistance marker transfected MSCs, and thendetected the efficiency of infection after48hours, transfected MSCs at MOI=200to determine the optimal concentration of lentivirus infection. qRT-PCRdetect the expression of miR-499, cardiac-specific genes GATA4, Nkx2.5, cTnIin MSCs after7days.2. After overexpressing miR-499in MSCs, qRT-PCR detected the expression ofcardiac-specific genes GATA4, Nkx2.5, cTnI mRNA.3. The models of acute myocardial infarction in SD rats were induced by leftanterior descending coronary artery ligation. After one week, according to themeans dealing with MSCs and the components injected into myocardium,Therats were randomly divided into5groups groups:①PBS,②MSCs,③SMHs,④miR-499MSCs,⑤SMHs+miR-499MSCs. 4.4weeks after cell transplantation, echocardiogram detected the cardiac function.5. After echocardiogram detecting, drawn immediately and hearts were frozen inO.C.T. for frozen sections, then observed the rate of cell retention in infarctedmyocardium under an inverted fluorescence microscope, immunofluorescencestaining detected the expression of cTnT, Desmin and Cx-43in implantedMSCs.6. paraffin, CD31immunohistochemical staining calculated the number ofneovascularization in infarct area and infarct border zone, The sections werestained with Masson’s trichrome and observed myocardial fibrosis in each groupand calculated ventricular wall thickness and infarct size.Results1. MSCs selected by puromycin and stably expressed miR-499,and then qRT-PCRdetected the expression of miR-499, cardiac-specific gene GATA4,Nkx2.5,cTnIcompared with negative control, the former had a rise.2. Four weeks after cell transplantation, echocardiography detect cardiac functionshowed treatment group had improved to varying degrees, SMHs+miR-499MSCs group was most obvious, MSCs group, SMHs group, miR-499MSCsgroup followed.3. Frozen sections observed under inverted fluorescence microscope, SMHs+miR-499MSCs group indicated that the rate of cell retention significantlyincreased, MSCs group, miR-499MSCs group were fewer.4. Immunofluorescence staining showed that compared with MSCs group, theexpression of cardiac-specific protein cTnT, Desmin and Cx-43in SMHs+miR-499MSCs group was the strongest, miR-499MSCs group followed.5. Masson’s trichrome staining showed that, compared with the PBS group, themyocardial infarct size and wall thickening in SMHs+miR-499MSCs groupsignificantly reduced and fibrosis also decreased. there was a markedimprovement in MSCs group, SMHs group, miR-499MSCs group, but inferiorto SMHs+miR-499MSCs group. 6. CD31immunohistochemical staining showed, the number of neovascularizationin infarct zone of SMHs+miR-499MSCs group was most, but there was nosignificant difference in infarct border zone compared with PBS group.thenumber of neovascularization in infarct border zone of all other groups werealso increased, but There was no significant difference between each groups.Conclusion1. miR-499could induces bone marrow-derived mesenchymal stem cells (MSCs)differentiate into cardiomyocyte-like cells in vitro.2. miR-499in vivo can promote transplanted MSCs differentiation into myocardialcells and reduces myocardial fibrosis, promotes angiogenesis in infarcted areaand improves heart function after MI. The retention of MSCs carried by SMHstransplantation increased significantly;miR-499combinating with SMHs aremore significant to improve heart function.