Study Of Transplantation Of Bone Marrow Mesenchymal Stem Cells For Treatment Of Myocardial Infarction

Objective To observe the biological characteristics of MSCs. To study the mechanism of 5 azacytidine (5-aza) inducing MSCs's differentiation towards cardiocytes.Methods The long bone of rat lower limb was separated and the bone marrow cells were obtained by density gradient centrifugation. The bone marrow cells were cultured in low-glucose DMEM with 15% fetal bovine serum and MSCs were purified by adherence to the wall of flask. When cells become more than 80% confluence, the adherent cells were digested using 0.25% trypsin and began to sub culture and proliferation. 48 hours later, MSCs of 4 experimental groups were treated with 5-aza of different concentration for 24 hours. No inducer was added to the contrast group. The expression of α-sarcomeric actin , connexin-43 (Cx-43) and cardiac troponin T (cTnT) was detected by immunocytochemistry; The existence of MSCs differentiating towards cardiocytes was examined by RT-PCR.Results (1) The MSCs grew adhesively, appearing long spindle, polyhedraletc.MSCs were purified gradually with changing medium. The primaryculturing cells were able to sub culture after incubation for 10-12days.(2)MSCs were able to proliferate actively and grew faster after reseeding. Thegrowing peak was reached at the 6^th or 7^th day.(3)MSCs became larger and irregular after inducing with 5- aza.(4)The data from immunocytochemistry show weak expression ofα-sarcomeric actin , connexin-43 and cardiac troponin T in the groups of 5μMafter 2 weeks and 3 weeks incubation and the group of 10μM after 2 weeksincubation. And strong expression in the 10μM group after 3 weeks incubation.Most cells in 50μM group died and were discarded during culturing.There isno positive expression in other groups.(5) The data from RT-PCR show the appearance of the gene band same withthe target band in the groups treated with 5μM and 10μM 5- aza incubationfor 2 weeks and 3 weeks, which indicate the positive expression ofα-sarcomeric actin and cardiac troponin T in these groups. There is no targetband in other groups.Conclusion In vitro MCSs culture and differentiation is feasible. 15% fetalbovine serum can meet the need of MSCs's growth .MSCs have the potentialof multi-directional differentiation and can differentiate into cardiocytesunder suitable inducing conditions. 5-aza can induce MSCs's differentiatinginto cardiocytes. The concentration of 10uM 5-aza with 24 hours incubation isthe most suitable.Objective To observe the proliferation and differentiation of congeneric MSCs transplanting into of another rat.To study the effect of MSCs transplantation on myocardial infarction and heart function and its mechanism.To provide new seed cell for cell transplantation treating acute myocardial infarction.Methods Myocardial infarct adult rat model was set up by ligation of left anterior descending coronary artery (LAD) after breast-opening.2 weeks later, the breast was opened again. L-DMEM, PBS and MSCs with BrdU were transplanted into the periphery and central of infarcting myocardia in 3 experimental groups respectively. Ultrasonic examination was used to detect heart function before and after 4-week transplantation respectively. After the second ultrasonic examination, kill the rat and take out heart.The heart were dehydrated routinely, imbed into paraffins and were sliced into 5μm thick slices continuously. The changes of infarcting myocardia at cellular and histological level were examined using HE staining and immunohistochemistry.Results (1) The heart function of L-DMEM and PBS treated groups became worse, while that of MSCs transplantation group got better (p<0.05). (2)Transplanting MSCs with BrdU were found in infarcting myocardia of transplantation group(3)MSCs express proteins specific to cardiocytes after 4-week transplantation. Conclusion MSCs can survive and proliferate after transplanted intomyocardial infarction parts. Under the microenvironments of heart, MSCs can differentiated into cardiocytes. MSCs transplantation can repair infarcting myocardia and improve heart function. MScs is one kind of the seed cells for treating the acute myocardial infarction. Part 3Therapeutic effects of the combination of MSCs transplantation and atrovastatin on acute cardiac infarctionObjective To study the different effects of MSCs transplantation and the combination of MSCs transplantation and atrovastatin on acute cardiac infarction.To study the mechanism of Atrovastatin's effective on MSCs transplantation and cardiac infarction, providing the evidence for improving therapeutic effect of cell transplantation on cardiac infarction. Methods After setting up animal model of cardiac infarction, rats were divided into 4 groups. Contrast group: L-DMEM was injected into infarcting myocardia region. Atrovastatin group: L-DMEM was injected into infarcting myocardia region. 10 days after operation, the rats were fed with 10mg/Kg.d atrovastatin for 4 weeks. MSCs group: Marked MSCs were injected into 4 points in myocardiac infarct region at the concentration of 50μl/point (containing 2×10⁶ cells). Combination of MSCs and Atrovastatin group: Marked MSCs were injected into 4 points in myocardiac infarct region at the concentration of 50μl/point. 10 days after operation, the rats were fed with 10mg/Kg.d atrovastatin for 4 weeks. 4-week later, the heart function, left ventricular ejection fraction (EF%) and the fractional shortening (FS%) weredetected respectively using Ultrasonic examination. The right common carotid artery was separated. The left ventricular systole pressure (LVSP), left ventricular end diastole pressure (LVEDP), and the maximal rate of left ventricular pressure (+dp/dt_max) were examined. The morphological changes of myocardia at cellular and histological level were measured immunohistochemistry.Results (l)Compared with left ventricular volume, left ventricular ejection fraction and fractional shortening of Contrast group and atrovastatin group, those of MSCs group and combination of MSCs and Atrovastatin group increased obviously (p<0.05). Furthermore, those of combination of MSCs and Atrovastatin group increased more sharply than those of MSCs group. (2)The mobility of anterior wall of left ventricle in contrast group and atrovastatin group weakened while that in MSCs group and combination of MSCs and atrovastatin group got stronger. .Furthermore, that in combination of MSCs and atrovastatin group showed stronger than that in MSCs group. (3)Compared with LVSP and +dp/dt_max of contrast group and atrovastatin group, those of MSCs group and combination of MSCs and atrovastatin group increased obviously (p<0.05). Furthermore, those of combination of MSCs and Atrovastatin group increased more sharply than those of MSCs group (p<0.05). Compared with LVEDP of Contrast group and atrovastatin group, that of MSCs group and combination of MSCs and Atrovastatin group decreased obviously (p<0.05). Furthermore, that of combination of MSCs and Atrovastatin group decreased more sharply than that of MSCs group (p<0.05). (4)In the myocardiac infarct region of both contrast group and atrovastatin group, the myocardial fibrosis was obvious and cells arranged irregularly. In the myocardiac infarct region of MSCs group and combination of MSCs andatrovastatin group,the marked cells can be found arranging regularly, similar to the arrangement of myocardiocytes.(5)Most of the Brd U positive cells showed positive expression of TnT and connexin 43.Conclusion Atrovastatin can inhibit inflammation and the formation of platelet thrombus, improving endothelium's function and blood supply of myocardia in myocardiac infarct region on the other. Atrovastatin can make the survival microenvironment of transplanting cells better and promote MSCs differentiating towards cardiocytes.Treatment with the combination of MSCs transplantation and atrovastatin on acute myocardiac infarct can promote the therapeutic effects cooperatively and improve heart function in a better way. The therapeutic effect of combination of MSCs transplantation and atrovastatin is superior to that of MSCs transplantation only.