| Background and purpose:Acute myocardial infarction is a common disease in human heart disease. It seriously threat to human health because of high mortality and heart failure. Traditional treatments can only slow down or improve the course of myocardial infarction, but can not regenerate the necrosis of the myocardial cell.In recent years, many animal experiments and clinical pre-test confirmed that cell transplantation therapy could increase the number of functional Cardiocytes and improve heart function. Bone Marrow stromal stem cells (BMSCs) is considered the best ideal seed cells in cell transplantation because of its convenient gain, availability of a large number of cells in vitro, low immune rejection and without ethical issues,ect. This study was to investigate the method of isolating and culturing BMSCs in vitro. BMSCs were directionally differentiated into myocardial cells and investigate the cellular mechanism of BMSCs transplantation for the treatment of MI and provide theoretical basis for clinic use in future.Materials and Methods:(1) BMSCs were isolated and identified in vitro:Rat BMSCs were gained by washing bone marrow from4weeks old Wistar rat and seeded at a density of1×107cells/ml.24hours after seeding, medium was changed for the first time. When cells covered with80-90%of Corning. Then the subculture seeded at a density of2×105cells/ml. The3rd generation cell were identified the expression of surface antigens CD29, CD34, CD90by flow cytometry.(2) BMSCs were induced in vitro and induced cell were identified:48hours after seeding, the3rd generation cells were exposed to5-aza.Inducer concentration were divided to1μmol/L,5μmol/L,10μmol/L,20μmol/L,50μmol/L.24hours after adding5-aza, medium was changed to remove inducer. Then cells which induced by10umol/L and cultured for14,21days were immunochemical stained for identification the expression of actin and CX43.(3) Establish rat myocardial infarction (MI) models:The left anterior descending coronary artery was ligated to establish rat AMI models. We identified the rat AMI models with the electrocardiogram, pathologic biopsy, TTC dyeing and heart appearance changing.(4) Cells were marked before transplanted and labeled cells were transplanted into cardiac muscle:the third generation of BMSCs were cultured in medium containing50μg/ml DAPI. Cells were adjusted at the density of1×10’cells/50μL and were injected into the myocardial infarction area in1hour.(5) Assess the survival and differentiation of transplanted cells and the effect of cell transplantation for treatment of MI rats: Myocardial tissue sections were stained with immunofluorescence to find cells which glowing both the blue fluorescent and red fluorescent hair under fluorescence microscope. we evaluated therapy effect by the electrocardiogram, observation of histological sections and evaluation of capillary density changes.Results:(1)24-hour after seeding, we can find small amount of round adherent cells. Then they gradually grew into fibroblast-like and showed colony formation.12days after seeding cells can be generated for the first time. Subculture just adherent cells were round, and soon extended to the short spindle, long spindle and irregular polygons. Subculture of the cell growth curve similar to "S" shaped and the rapid appreciation of time was3-5days and the plateau was7days. The test result from FCM indicated that the positive rate of CD29、CD34and CD90are84.8%、3.2%and95.3%, and they conform to the characters of BMSCs.(2) We knew the suitable dose of inducer is10μmol/L5-aza from comparing the induction results and cell shape is similar to cardiomyocytes. Immunocytochemistry showed us that induced cells express the protein of Actin and CX43. BMSCs differentiated into cardiocyte cells by5-aza.(3)The evaluation of MI rat model:Compared to control group rats, the ECG of experimental group appearred Q wave abnormalities and/or ST-segment elevation (0.2-0.4mV) and/or T wave inversion. In addition, we observed MI myocardial tissue sections and found that the basic structure of myocardial was disorder and nuclear become condensation and stained deepened and some nuclear melt away. TTC staining showed that infarction tissue was white, but normal tissue was dark red. All those proved the successful of MI model and the success rate was65.51%.(4)3weeks after cell transplantation,we assessed the preliminary treatment of AMI in rats:ECG showed us it returned to basic normal. HE stain of pathological tissue sections appeared a larger proportion of cells nucleus-to-cytoplasm ratio, and the growth orientation of those cells lined up with the direction of the muscle fiber cells. Capillary density in the experimental group was larger than the control group and it improved the blood supply to the heart. The Immunofluorescence detection of cell transplantation group showed there were cells glowing the blue fluorescent and red fluorescent, which indicated that transplantation BMSCs survived in the infarction area and directionally differentiated into myocardial cells.Conclusions:Rat BMSCs were suitable for isolation and amplification and induction; BMSCs can be induced differentiation into cardiocyte cells by5-aza and expressed the cardiac proteins of actin and CX43; we can establish myocardial infarction model by ligating the left anterior descending coronary artery;3weeks after transplantation, BMSCs can survive in myocardial infarction parts and differentiate into cardiocyte cells and improve cardiac function in rats. |
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