Adrenomedullin Gene Transfection Enhances The Therapeutic Effects Of Transplantation Of Bone Marrow Mesenchymal Stem Cells On Ventricle Remodeling And Cardiac Function In Acute Myocardial Infarction Rats

PartⅠ: The experiment of Ad-ADM transfected MSCs in vitroObjective: To investigate the transfection efficiency of mesenchymal stem cells by Ad-LacZ and the expression of ADM in MSCs infected with Ad-ADM. Methods: MSCs were isolated and expanded using the preplating method. The infection efficiency of Ad-ADM to MSCs was tested by x-gal stain. Ad-ADM expression in MSCs and its secretion in culture medium were measured by ELISA.Results: (1) MSCs can be successfully isolated and cultured by adhesion method.The passage cells of MSCs had gradually shown homogeneity, spindle-like, and radiative. (2)The teter of Ad-LacZ, Ad-ADM was 2.8×109pfu/ml, 1.1×109 pfu/ml respectively. (3) With x-gal stain, MSCs could be effectively transfected by adenovirus in vitro, the transfection efficiency has the dose-effect relationship with multiplicities of infection (MOI) . When MOI was 150, the infection efficiency was 95.4%. The expression of ADM was traced in culture medium and has the time-effect relationship . A maximum production of ADM was observed at 7 day after infection (pg/ml: 26.53±1.42 VS 1.34±0.075, n=3,P<0.05),and ADM secretion reduces to control level in 15 day (pg/ml:2.2±1.44 VS 1.52±0.33,n=3,P>0.05).RT-PCR showed the same result. Conclusions:①MSCs could be effectively transfected by adenovirus in vitro.②The expression of ADM was traced in culture medium and has the time-effect relationship. PartⅡ: Effects of transplantation of allogenic MSCs transfected by ADM gene on the treatment of myocardial infarction in ratsObjective: To study the Effects of transplantation of allogenic MSCs transfected by ADM gene on the treatment of myocardial infarction in rats. Methods: The left anterior descending branch of rats was ligated to establish a myocardial infarction model. Then the MSCs were labeled by DAPI, and were directly implanted into the acute infarct site via focal injection. 61 Male SD rats were randomly divided into five group:acute myocardial infarction injected DMEM group as control (control,A group,n=12),myocardial infarction puls Ad-ADM group(Ad-ADM ,B group,n=12), myocardial infarction puls MSCs transplantation group(MSCs,C group,n=12), myocardial infarction puls ADM-transfer -MSCs transplantation group(Ad-ADM+MSCs,D group,n=14) and Sham group as E group(n=11). Four weeks later, cardiac function was evaluated using physiological recorder and echocardiography. Hearts were harvested. Sirius red staining was used to identify interstitial collagen on slides. Analysis of collagen typeⅠandⅢwas performed using a polarized filter on sections stained for collagen with Sirius Red, and the ratio of collagen type I and III were detected. Fluorescence microscope was used to identify the DAPI-labeled cells. Immunohistochemistry staining of factorⅧwas used to evaluate the density of capillary vessels in infarction area. Immunohistochemistry staining of Troponin I (TNI), connexin43 and ADM were used to evaluate the differentiation and expression of implanted MSCs. Results: Immunohistochemical studies demonstrated that intense immunostaining for ADM was higher in Ad-ADM plus MSCs group, compared with other group. Compared with control, MSCs transplantation significantly increased capillary density in infarct area (P<0.01). MSCs transplantation decreased the ratio of collagen typeⅠandⅢ, then obviously improved left ventricle functions. Furthermore combination group (D group) resulted in further decreases the ratio of collagen typeⅠandⅢ, and significantly improved left ventricle functions. DAPI-labeled transplantion MSCs were founded in the hearts of the recipients and expressed TNI in all MSCs transplantation groups(C and D group). Immunohistochemical studies demonstrated that intense immunostaining for connexin 43 was higher in Ad-ADM plus MSCs group, compared with other group. Compared with control, MSCs transplantation significantly increased capillary density in infarct area(P<0.01),and obviously improved left ventricular ejection fraction(LVEF). A combination of Ad-ADM trensfection and MSCs transplantation demonstrated a further increase in capillary density and further improved LVEF compared with MSCs alone. Conclusions Ad-ADM transfection enhanced the angiogenic potency of MSCs transplantation, decreased the ratio of collagen typeⅠandⅢ, and improved cardiac function. ADM gene transfection did not affect differentiation of MSCs into cardiomyocyte-like cells in vivo.