| Research surroundings:Acute myocardial infarction is a familiar and common disease that severely threatens the elderly's health. At the present time, the ultimate treatment to regenerate the infarcted myocardium is lacking. Although thrombolysis and PCI in 4-6 hours after onset of myocardial infarction can improve the blood perfusion in the infarct-related vessels and save the dying cardiomyocytes, the reperfusion therapy only can limit the infarcted size and have no effect on the infarcted myocardium. Lacking the ability of differentiation and regeration, the infarcted myocardium can only be replaced by the fibroblasts and form the insystolic scar tissue. Thus the patients are easy to suffer from heart failure, even sudden cardic death, and their life quality is also very poor. The cellular transplantation technique gives us a good prevision to regenerate the injured myocardium. Fetal cardiomyocytes, embryonic stem cells, skeletal myoblasts, bone marrow stem cells became the focus of this field in present times. The fetal cardiomyocytes is difficult to be obtained and the embryonic stem cells have the ethical, legal problems and immune rejection. So it'sdifficult for them to use in clinic. Skeletal myocyte is also leiomyoctes as cardiomyocytes, transplanting skeletal myoblasts to regenerate the injured cardiomyocytes have made some progress. But the quantity of skeletal myoblasts is limited, just about 4%-8% of the total skeletal myocytes, and also decrease with age. The bone marrow stem cells are the multipotent adult stem cells and can be used for autologous transplantation. But it still has the disadvantage of less quantity and is difficult to be obtained. It's indicated recently that the adipose tissue-derived mesenchymal stem cells (ADMSCs) also have the multipotent differentiation ability. At appropriate conditions, these cells can differentiate into osteoblasts, adipose cells, nerve cells, cardiomyogenic cells et al. In addition, ADMSCs are easy to separate and cultivate, have the strong ability to reproduce in vitro. All these characters make ADMSCs become the focus of the cardiology fields and become the ideal optional therapeutic method to regenerate the cardiomyicytes after the myocardial infarction.Objective:(D to establish a method for isolation, purification and cultivation of ADMSCs; (2)to observe if ADMSCs can differentiate into cardiomyocytes in vitro; (3) to observe the migration and differentiation of the rat ADMSCs in infarcted heart tissue; ?to observe the therapeutic effect of the homogeneously exogenous rat's ADMSCs transplantation cure rat myocardial infarction. Our final perpose is to provide a new thought, a new way and a new method to cure myocardial infarction and to lay a foundation of clinical use for ADMSCs.Methods and results:1. To explore a method for isolation and cultivation of mesenchymal stem cells from adipose tissue in vitro.The adipose tissue was obtained from the epiploon of rats under the aseptic condition. The fat then was minced and digested with 5ml 0.08% for 5minutes with constant agitation. The monocytes layer was sucked out and new trypin was addedinto the remnant. The process above was repeated for 3 times. Then the collected cells were supplemented with albumin, cultured in IMDM with 15% fetal bovine serum. Immunocytochemistry was used to test the surface molecule CD13,CD44, CD45 and HLA-DR. CD Band CD44 were the surface molecule to detect the MSCs, CD45 to detect the hematogenic stem cells and HLA-DR to detect the fibrobasts. Immunocytochemical staining shows that the 90% obtained cells are the CD13,CD44 positive MSCs and the CD45, HLA-DR negetive. Thus we establish a simple and convenient method to separate and culture the adipose tissue-derived mesenchymal stem cells and provide the reference for ADMSCs actual application.2. to establish the rat acute myocardial infarction modelLeft coronary artery of rats were legated to cause acute myocardial infarction, and the ECG changed at once. The ST segment of Vi~V6 rose and that of II ,111, avF depressed. 28 days after operation, the ultrasonic cardiac graph and the hemodynamics both show that the heart function of the AMI group decreased compared with the sham group. The pathological detection show that the scars were seen on the left ventricular that make the left ventricular wall thin and sunken, and the normal cardiomyocytes disappeared and were taken place of connective tissue under the microscope. Thus we establish the rat's myocardial infarction model successfully.3. To establish a method of inducing the ADMSCs into cardiomyocytes.The third passage cells were used to induce into cardiomyocytes .The cells were cultured with 1 Oumol/L 5-AZA for 24hours. Then the medium was changed and the cells were washed to get rid of the remained 5-AZA. The cells were continued to culture for 3 weeks and observed the changes. One week after inducing, the shape of part of shuttle-like cells became ball-like or ovum-like. Two weeks after inducing, the ball-like cells began to congregate and the bulk became large. Three weeks after inducing, the cells continued congregating and formed the cell group, at the same time the cardiomyocytes specific antigen tropninl, a -actnin and desmin became positive expressed. The mesenchymal stem cells obtained from the adipose tissue candifferentiate into cardiomyocytes induced by 5-AZA.4. To investigate the effect and possibility of exogenous transplantion of the adipose tissue derived mesenchymal stem cells for treatment of rat acute myocardial infarction. 18 male rats were separated randomly into 3 groups: sham surgery group (control n=6), acute myocardial infarction control group (AMI n=6) and myocardial infarction plus cell transplantation group (AMI+cell n=6). The infarcted hearts were made by occlusion of left coronary artery. The mesenchymal stem cells were isolated from the rats' peritoneum using digestion methods and reproduced in vitro. Then the cells were labeled by BrdU and implanted into the infarcted heart of the rats. Heart functions were measured 4 weeks after implantation. The hearts were also harvested for pathological observation and histoimmunochemistry to detect the survivance and location of the implanted cells. Compared with the AMI group, the left ventricular systolic pressure of the cell therapy group was increased significantly (P<0.01),the left ventricular end-diastolic pressure was decreased (P<0.01) and the ratio of the left ventricular pressure rise and decay(±dp/dt) was decreased (P<0.05). The number of blood vessels were increased at the boundary of infarction site by pathological observation. The labeled cells were founded in the infarcted myocardiocytes and the blood vessel wall. The methods of exogenous transplantation of adipose tissue derived mesenchymal stem cells for treatment of AMI is effective and feasible.Brief summary:1. The adipose tissue contained plenty of stem cells. When ADMSCs syncretized 70%-80%, seeding in time can avoid self- differentiation and also is the main method to purify the ADMSCs.2. ADMSCs can be induced into cardiomyogenic cells by 5-AZA in vitro.3. The exogenous ADMSCs have the ability to migrate into the ischemic andinfarcted regions and differentiate into myocardium-like cells and take part in the growth of new vessels.4. Transplanting the ADMSCs can improve the heart function of infarcted rat heart.Conclusion :Though the research of separating, culturing and inducing the ADMSCs in vitro and transplanting the ADMSCs into the rat's infarcted cardiomyocytes in vivo, we provide the experimental data for a new stem cell source that is easier to obtained, more acceptable for patients and have effective effects. And we also cut out a new fields for ADMSCs to prevent and cure the myocardial infarction. |
PDF Full Text Request