| Objective:Wide attention has been paid to the therapeutic strategy of cell transplantation which improves myocardial infarction through cell differentiation, angiogenesis and other mechanisms in recent years. However, only a very small number of transplanted cells can survive after the transplantation. Therefore,it is important to study the factors that influence the survival rate of transplanted cells.In this experiment, bone marrow-derived mesenchymal stem cells which were isolated from the male SD rats were transplanted into the female rats which were suffering from acute myocardial infarction.We investigated the dynamic state and distribution of the surviving rat mesenchymal stem cells engrafted in infarcted heart firstly. We hypothesis that the high concentration of NO is one of major contributors to the apoptosis of transplanted cells after myocardial infarction. So,some more work was made to study the changing expression of induced nitric oxide synthase (iNOS) after myocardial infarction.On this basis,we analysed the protected effect on the further survival rate of the engrafted mesenchymal stem cells after giving iNOS selective inhibitor 1400W.Methods:(1) Female SD rats weighing 150-180g were prepared to make acute myocardial infarction model. Acute myocardial infarction (AMI) was created by occluding the left anterior descending artery (LAD); The cells were transplanted 1 h after AMI and the animal models were randomly divided into several groups and detected. (2) BMSCs were isolated from the male SD rats weighing 100-130g, purified, proliferated and then they were detected by flow cytometry and immunohistochemical detection. (3) 1.2×106 BMSCs were injected into the centre zone of the infarct region at 1 h after MI. (4) at 1d,3d,7d,10d and 14d after AMI,the infarct regions were harvested and the expression of iNOS was analysed by western blot.(5) The BMSCs were labeled with Brdu or hoechst33342 previously and after BMSCs transplantation,the infarct regions were harvested and observed by fluorescence microscope or studied by HE staining and Brdu immunohistochemical examinations. (6)A Sry suquence of male rats Y chromatosome was analysed by the real time polymerase chain reaction.The results:1. After the anterior descending artery ligation, heart ischemic area became pale,the pulse was weakened and clear boundary appeared compared with no ischemia area; After isolation and purification, MSCs showed homogenous with CD44+/CD34-.Immunohistochemical detection demonstrated CD44 was positive, CD34 was negative and proved that the experimental cells were BMSCs.2. The dynamic state of the surviving cells engrafted in the infarcted heart:The cells labeled with hoechst33342 in different groups were viable in large numbers in the host hearts and a small quantity of them migrated from the center to the margin of the infarct region. Immunohistochemical staining revealed that Brdu-labelled BMSCs with oval nucleus were localized. The real time polymerase chain reaction demonstrated that the survival rate of engrafted BMSCs in three groups were 7.88%(1 week group),7.82%(3 weeks group),8.73%(6 weeks group).The result demonstrated that from one to six weeks there were no progressive decrease of the transplanted cells.3. Western blot detected the expression of iNOS: Different time myocardial infarction changed the intensity of iNOS expression:three days> 1 day> 7 days> 10 days> 14 days (n= 6, p<0.05);4. iNOS selective inhibitor 1400W contributed to further survival rate of the transplantated cells:The cells labeled with hoechst33342 in the test group and control group were both viable in large numbers in the host heart and a small quantity of them migrated from the center to the margin of the infarct region. Compared with control group,the cell density were higher in the test group. Immunohistochemical staining revealed that Brdu-labelled MSCs with oval nucleus were localized in both of the groups. The real time polymerase chain reaction demonstrated that the survival rate of engrafted BMSCs in two groups were 8.75%(the test roup),6.63% (the control group);The survival rate of the engrafted cells in test group were significantly higher than the control group (n= 7, p<0.05).Conclusion:1. From one to six weeks after cell transplantation, BMSCs could survive stably in the host infarcted heart and there were no progressive decrease. The cells were localized firstly and later they migrated from the center to the margin of the infarct region.2.The expression peak of iNOS is the 1-3 day after myocardial infarction.3. iNOS selective inhibitor 1400w can significantly improved the further survival rate of transplanted cells.
|
PDF Full Text Request