| Objective: Acute myocardial infarction as a common cardiovascular disease, is a serious threat to human's life. Many studies indicate that bone marrow stem cell transplantation for myocardial infarction, can significantly repair damaged myocardium, induce angiogenesis and improve heart function .But there are still a large number of issues limiting its clinical application. For example, only a very small number of transplanted cells can survive after the transplantation or right timing for transplantation. After myocardial infarction, myocardial ischemia and hypoxia, a large number of harmful cytokines and inflammatory mediators are released, inducible nitric oxide synthase (iNOS) activity is activated, may lead a large number of transplanted cells to death. Therefore, it is important to study the factors that influence the survival rate of transplanted MSCs . In this experiment, bone marrow mesenchymal stem cells which were isolated from the male SD rats were transplanted into the female rats which were suffering from acute myocardial infarction in different time points after myocardial infarction.We observed the survival rate and distribution of transplanted cells and heart function, explored the best time for transplanting and studied the changing expression of iNOS after myocardial infarction. On this basis, We observed the survival rate of transplanted cells and cardiac function by intervention of iNOS selective inhibitor 1400W, identified its roles in cardiac repair.Methods: 1. Female SD rats weight of 150-180g were prepared to make acute myocardial infarction model. Acute myocardial infarction was created by occluding the left anterior descending artery, the rat models were randomly divided into several groups and detected according to the different times of transplantation.2. MSCs were isolated from the male SD rats weight of 100-130g through bone marrow adherent,purified ,proliferated and then detected by flow cytometry and immunocytochemistry.3. About 1.2×106 MSCs were injected into the centre zone of the infarct region at different time after MI.4. MSCs were labeled with Hoechst33342 and BrdU previously. After transplantation, the infarct regions were harvested and observed by fluorescence microscope or studied by HE staining or immunohistochemical examinations, then observed the location and distribution of transplanted cells in the infarct area.5. Sry suquence of male rats Y chromatosome was analysed by Real-time PCR as a quantitative comparison of indicators of survival.6.Giving cardiac echocardiography examination after 4 weeks. 7. The expression of iNOS in the infarct regions was detected by Western-blot at 1h,3d,7d,10d and 14d after myocardial infarction.Results: 1. The fourth generation of MSCs have been detected by immunocyto- chemistry expressed CD29 and CD44, no CD34 expression. Flow cytometry analyzed CD44 + / CD34 -, proved that the experimental cells were MSCs.2. After coronary artery ligation, left ventricular anterior wall of pale, heart enlargement, left atrial appendage congestion, ventricular contraction decreased, with no clear lines of infarct area, indicating that the animal model was successful.3.Rats were randomly divided into 4 groups, myocardial infarction (MI) group: Myocardial infarction without MSCs transplantation, giving cardiac echocardiography examination after 4 weeks as a control; 1h transplantation group: MSCs were transplanted at 1h after myocardial infarction, sry genes of Y chromosome were detected as the control specimens; 3d transplant group: MSCs were transplanted at 3d after myocardial infarction; 7d transplant group: MSCs were transplanted at 7d after myocardial infarction. 3d and 7d groups were both given cardiac function test, survival rate analysis of transplanted cells and tissue testing after 4 weeks. The survival rate of transplanted cells of 7d group was significantly higher than the 3d group by Real-time PCR detection, MSCs survival rate was 8.1%, 2.5% (n = 10, p <0.05). After 4 weeks cardiac function tests found that compared with the MI group, left ventricular end diastolic diameter (LVDd) and end systolic diameter (LVDs) of 3d group were no significant narrowing, left ventricular fractional shortening (FS) and Left ventricular ejection fraction (EF) was also not significantly increased(n = 10, p> 0.05). LVDd and LVDs of 7d group were significantly reduced than 3d and MI group, FS and EF was significantly improved, there was much more significant difference (n = 10 , P <0.05). Histology detection found that Hoechst33342 and BrdU-positive cells were mainly distributed in the graft area in 1h group; there were a little Hoechst33342 of BrdU-positive cells in the infarct regions in 3d group; On the contrary, a large number of transplanted cells with blue fluorescence distribution in the infarct area in 7d group, some cells aggregate to form blood vessel-like structure. BrdU immunohistochemical staining and the corresponding HE staining had the same consequence, suggesting that transplanted MSCs were survived, proliferated in the host cardiac tissue, and may be involved in formation of new blood vessels, promote cardiac tissue repair.4. The expression of iNOS in the infarct myocardium immediately increased after myocardial infarction, peaked in 3 days then gradually decreased.The intensity of iNOS expression at different time points after MI is: 3d> 1h> 7d> 10d> 14d (n = 5, p <0.05).5. The selective iNOS inhibitor 1400W was given ahead 12h of transplantation in the treatment group 2mg/kg, intraperitoneal injection, 2 / day, maintained for 3 days, control group with normal saline instead. Real-time PCR analysis showed that sry gene expression levels of Y chromosome in 1400W treatment group and control group were 4.2%, 1.8% (n = 10, P <0.01). Histological examination showed that BrdU-positive cells were scattered, few in infarct area in the control group; but in the treatment group BrdU-positive cells were significantly increased and more concentrated. HE staining had the similar results. However, heart function test in the treatment group and control group showed no significant difference (n = 10, p> 0.05). Experimental results show that the early intervention of iNOS inhibitors can improve the survival rate of transplanted cells of 3d transplantation group, but has no help to the improvement of cardiac function.Conclusion: 1. Concerning the survival rate and cardiac function,cell transplantation at the 7th day after myocardial infarction is superior to the transplantation at the 3th day.2. iNOS expression peaks at 3 days after myocardial infarction, and then decreases gradually.3. The early intervention of iNOS inhibitors can improve the survival rate of transplanted cells of 3d transplantation group, but not enough to cause the changes in heart function.
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