| Background and ObjectiveAcute myocardial infarction (AMI) has been a leading cause of morbidity and mortality in human, but at present how to prevent and treat it is still a difficult problem. With the development of study on stem cells, many evidences have indicated that marrow stem cells could differentiate into cardiomyocyte in vivo and in vitro. This brings us the hope of regenerating cardiomyocytes and restoring damaged hearts. Studies suggested that mesenchymal stem cells (MSCs) had the capability of recruiting into injured organs and repairing them and this characteristic of MSCs implied the validity of delivering MSCs through vein to treat injured organs. While many studies simultaneously indicated that the ability of MSCs recruiting forward and rebuilding injured organs were limited by several conditions, such as the new injuries of organs and the number of homing MSCs. However, although there were great deals of experiments probing into the differentiating capability of MSCs, few investigations had discussed the transplantation time, especially through blood vessel route. This studyinvestigated whether MSCs delivered intravenously had the encouraging ability of migrating into infarcted heart of rat, and whether they could improve injured cardiac function; and discussed the effective transplantation time simultaneously. MethodsMSCs were isolated from bone marrow aspirates of isogenic SD rats, cultured and expanded ex vivo. To detect the transplanted cells, MSCs were prelabeled with bromodeoxyuridine (BrdU). SD rats were subjected to AMI by left coronary artery anterior descending branch occlusion. 88 AMI rats were divided into groupI (AMI+MSCs): I A, I B, I C, I D, I E (n=8); group II (AMI+PBS): II A, IIB, IIC,II D, IIE (n=8); and group III(only AMI, n=8). In addition, another 10 SD rats were only subjected to opening breast and traversing the thread without ligating coronary artery, as sham-AMI group (group IV), and then randomly divided into five subgroups: IVA,IVB,IVC,rVD,IVE (n=2). Five subgroups in three groups respectively represented different transplantation time points (lday and 1, 2, 3, 4weeks after surgery). GroupI and IV were given BrdU-labeled MSCs(l X 107), and group II were given equal volume of PBS through tail vein. To evaluate the cardiac function, all rats were performed echocardiography examination to calculate fractional shortening (FS) and left ventricular ejection fraction (LVEF). Group I and II received echocardiography examination at 1 day after surgery and 3 weeks after MSCs or PBS injection respectively; group III received it at 1 day and 3, 4, 5 , 6, 7weeks after AMI, but group IV were only examined at 1 day after surgery. Then, at the corresponding time points, all rats were killed and the hearts were obtained to perform immunohistochemical analysis. Slides were stained with hematoxylin and eosin (HE) and specific antibodies. Anti-BrdU and anti-cardiac troponin T (cTn-T) were used to identify the donor cells and the cardiac muscle respectively. Results(1) Preparing of transplant cells: After 24 hours, some round cells presented adhering to the bottom of culture plate, and through exchanging culture medium, the suspending cells were deleted. After 48 to 72 hours, the round cells adhering to thebottom began to transfiguring, and showed fibroblast-like. About 10 to 12 days, cells could cover all over the bottom, and then were released and passaged. When cells were co-cultured with BrdU for 48 hours, these cells would be labeled, with the positive ratio at about 75 percent.(2) Survival and death of experimental rats: There were no deaths among group I A, IB, I C, II A, II B, IIC and IV during the period of experiment; 3 rats died in group I D and III respectively; 4 rats died in group IE, II D and IIE. And all the deaths occurred during 6 to 7 weeks after surgery.(3) Echocardiography assessment: One day after surgery, except sham-AMI group, there were no differences in FS and LVEF among other groups (P>0.05) . Contrasting with one day after AMI, cardiac function (FS and LVEF) of group I A andI B presented significant improvements 3 weeks after cells transplantation (jP<0.001, respectively). On the other hand, contrasting with PBS groups at the same transfusion time point respectively, group I A, IB had significant improvements in cardiac function (FS and LVEF) 3 weeks after cells transfusion (F<0.01, respectively), while group I C had significant difference only in FS (P<0.01) . In addition, II A, II B andII C had no significant differences in cardiac function compared with group III at the same time points (.P>0.05, respectively).Because of the high death rate during 6 to 7 weeks after AMI, all groups were performed echocardiography examination only during 5 weeks after AMI.(4) lmmunohisroehemical analysis: HE stain showed the scar fiber tissue in all specimens except sham-AMI rats. Only slides of group I A, I B and I C displayed positive to staining with anti-BrdU. In addition, anti-BrdU stain positive cells expressed Tropnin-T.Conclusion1. MSCs of rats are easy to be isolated, cultured and expanded; ligating anterior descending branch of left coronary artery to make AMI model of rat is feasible.2. At the early stage of AMI (1 to 14 days), MSCs delivered intravenously can home into injured hearts, survive there, and may express cTn-T.3. MSCs homing into infarcted regions can improve cardiac function of AMI rats; this function may partly contribute to differentiating into cardiomyocyte-like cells.
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