| Objectives:Myocardial infarction (MI) is a common disease that severely threatens the elder's health. At the present, the therapeutic methods on MI including drugs, interventional therapy and so on are considered to have some curative effects on MI. But all these methods can't replace the necrotic myocardium. Cell transplantation therapy of myocardial infarction is an ideal one which can increase the population of functional contractile cells and improve the cardiac function. This therapeutic method has been considered to be a general application in clinical field. Now,Many scientists consider that the skeletal myoblast is an ideal donor cell, but most results come from the research of the primary myoblast. Our research tries to translate the immortalized L6 skeletal myoblast into the myocardial infarction field. The feasibility and the therapeutic effects in cardiac function with different quantity of L6 myoblast will be studied and it will provide the date of stem cell therapy for treating myocardial damage in basical research.Methods1 Culture and labeling methods of L6 myoblastAfter resuscitation, the rat skeletal myoblast cell line L6 was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and antibiotic. (under the condition of 37℃,5% CO2). Passage the cells when it syncretize 80%~90%. The L6 cells of log phase were labeled by colloidal gold for 20~24 hours, and then they were divided into different tubes for transplantation. Observe the cell appearance and maker rate by hematoxylin eosins (HE) staining and transmission electron microscope (TEM).2 Making the rat's myocardial infarction model and assessing the change of cardiac structure after infarction60 male healthy SD rats were chosen for the myocardial infarction model. We use the HE staining and the immunohistochemistry staining to assess the change of cardiac structure after left anterior descending coronary artery ligation at 1 day, 1 week, 2 weeks, 3 weeks and 4 weeks after infarction. With the method of flow cytometry (FCM), the cell cycle was examined.3 Study on development of L6 myoblast after transplantation of rat in vivo95 male healthy SD rats, were randomly divided into five group:control group, 1×106 group, 2×106 group, 4×106 group and 1×107 group. The control group has 15 rats; other group has 20 rats respectively. After left anterior descending coronary artery ligation, L6 myoblasts were transplanted into the center of myocardial infarction respectively. The control group was injected with the same volume non-serum DMEM. We use the methods which include the HE staning, immunohistochemical staining, TEM and FCM to assess the proliferation and differentiation of the implanted cells after MI. At 4 weeks after transplantation, the hemodynamics index was detected to assess if the implanted cells can improve the cardiac function.Results1 Culture and labeling results of L6 myoblastL6 myoblasts grow well at 1 day after seeded and most of them are fusiform shape. There have 1 or 2 oval-shap nucleus in the centre of the cells. The culture cells proliferated so fast that they can syncretize 80%~90% during 2 days. The colloidal gold labeling method is very well, we can see the colloidal gold particles in the endoplasma by TEM, the labeling rate is at least 80%。2 The change of cardiac structure after infarctionHistological analysis shows that the cardiomyocytes necrosis is palpable at 1 day after MI and most of them are coagulation necrosis. The pervasion of inflamed cells are gradually obvious at 1 week, the myofibers are arrayed out of order or broken up in part of the myocardium. After 2 weeks, fibroblast activation can be detected and the myocardium began to replace by scar tissue. With the development of fibrosis, it can form penetrating infarct area in left cardiac ventricle at 4 weeks. Immunohistochemistry and FCM results suggest that the cardiomyocytes can proliferation in earlier period after MI, but this capability is very limited and it will be weaken as the time go on.3 The development of L6 myoblast after transplantation of rat in vivoWe assess the development and differentiation of L6 myoblast after transplantation by the immunohistochemical staining, TEM, FCM and so on. Immunohistochemical staining results: There are a large number of PCNA positive L6 cells in early period after implanted, but the quantity is reduced by time. Desmin expression is obviously at 2 weeks, we can see some myotubes in the graft cells. Cx43 and GATA-4 are negative all the time. These results suggest that L6 myoblasts can survive and proliferate in the infarct area. Some of them can differentiate into myotubes, but the implanted cells don't communicate with the surrounding cardiomyocytes and differentiate into myocardium.Compare with the control group and the 1×106 group, LVSP, LVdp/dt and LV-dp/dt rising significantly but the LVEDP keep declining. The differences between 1×106 group and control group have no significance. The cardiac function results at 4 weeks after transplantation suggest that the L6 myoblast transplantation can improve the cardiac function of MI rats and more cells lead to better effect, but not the much the better. We find tumor-liked grafts in 4×106 and 1×107 group, the graft cells proliferated so extensively that they invaded and replaced surrounding myocardium. The ultrastructural study show that the nucleu of graft cell is become larger, moreover, the proportion of nucleus to plasm is more than 1:3, some nucleus have obviously heteromorphism. DNA value by FCM show that the DNA index (DI) of the graft cell is 1.31±0.14, it has significant difference compare with the normal group (DI=1.00±0.11). All the results above imply that too much L6 myoblasts has tumorigenicity.Conclusion1 Coronary artery ligation myocardial infarction model is a better model in studying myocardial repair because it has the similar organizational changes with the clinical situation and a lower mortality rate.2 The survival normal myocardial cell near/in the infarction area can proliferate after myocardial infarction.3 Colloidal gold labeling method has a better effect in vitro but not satisfactory in vivo. Implanted cells can not be recognized easily under LM and TEM. We should do more work to find a better labeling method for transplanted cells.4 The L6 myoblast can survive and differentiate in the center of heart infarction after transplantation. The proliferation ability is much stronger than the differentiation ability.5 Contrast with 1×106 , 2×106 L6 skeletal myoblasts have better effect in improving cardiac function .6 Too much L6 myoblast may not better, the grafts proliferated so extensively that they invaded and replaced surrounding myocardium, some times distorting the ventricular contour.
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