Allogeneic Bone Marrow Mononuclear Cell Transplantation In The Treatment Of Rats With Acute Myocardial Infarction Study

Part Two ISOLATION,CULTURE,AND PROLIFERATION OF RATS BONE MARROW MONONUCLEAR CELLS IN VITROObjective;To investigate the optimal condition for isolation,culture and proliferation of bone marrow mononuclear cells(BMMNCs),and observe the growth and biology characterization of BMMNCS.Methods;BMMNCs were isolated from adult rats bone marrow using density gradient centrifugation,and were cultured in low-glucose DMEM supplement with 15%FBS for expanding.Then bone marrow mesenchymal stem cells(BMMSCs) were obtained and purified via adherence culturing,.The morphology and characterization of BMMNCs and BMMSCs were observed at different time.Results;Colonies of BMMSCs formed at 4th day from primary culture of BMMNCs.From 5th to 12th day,BMMSCs grew faster and got together to form bigger colones then before.Afer 14 days from the beginning,BMMSCs fused in monolayer;then were subcultured by 0.25%trypsine-0.02%EDTA.The passage BMMSCs proliferated fast,and could be subculture about every 5 days.In later generation,BMMSCs grew low,and mutlipled karyotype appeared.Conclusion;BMMNCs obtained via density gradient centrifugation contained many BMMSCs;BMMSCs could be purified primarily via adherence culturing from BMMNCs. Part Two STUDY ON ESTABLISHMENT OF ACUTE MYOCARDIAL INFARCTION MODEL IN RATSObjective;To develop a steady and repeatable myocardial infarction model for later BMMNCs transplangtation in rats.Methods;20 Wistar rats were divided randomly into two groups.Afer the rats were anaesthetized with sodium phenobarbital and ventilated,left anterior descending coronary arteries were ligated in experimental group.Electrocardiogram(ECG) changes were observed before and immediately,30 minutes,1 hour after ligation.Echocardiographies were performed before and 8 weeks after operation. Pathologies were examined after 8-week echocardiography post-infarction.Results;1.ST segments onⅠ,Ⅱ,Ⅲ,aVF leads elevated persistently after ligation of LAD.2.Echocardiography manifested that;all parameters were concordant between control group and experimental group before operation,p＞0.05;8 weeks later, LVEDd and LVESd in experimental group increased 42.9%and 82.5%compared with control group,p＜0.001 respectively;end-systolic anterior wall thickening ratio(ESAWTR)in experimental group reduced significantly compared to control group,p＜0.001;end-systolic posterior wall thickening ratio(ESPWTR)increased significantly compared to control group,p＜0.01;left ventricular ejection fraction(LVEF)in experimental group decreased significantly compared to control group,p＜0.001.3.HE and Masson staining manifested that the cardiac tissue in ischemic area was replaced with fibrous connective tissue.Conclusions; 1.Ligation of LAD cause large area of myocardial infarction in anterior,lateral and inferior wall of rats heart.2.ST segments elevated more than 4mm onⅠ,Ⅱ,ⅢⅢ,aVF leads can predict large area of myocardial infarction in anterior wall of rat heart.3.Echocardiography is a good method to be used to observe the morphology and function of rat heart after myocardial infarction. Objective;To explore the long-term effects of bone marrow mononuclear cell transplantation on cardiac function,its mechanism and optimal time for cell transplantation in rats.Methods;36 adult inbred male Wistar rats were divided randomly into 3 groups;control group(no transplantation of BMMNCs),n=10;group of transplantation of BMMNCs immediately after AMI,n=10;group of transplantation of BMMNCs 1 week after AMI,n=10;the rest 6 inbred Wistar rats were used as BMMNCs donor to simulate autologous transplantation of BMMNCs.Before cell transplantation, BMMNCs were separated from bone marrow as before,and labeled with DAPI. Fluorescence microscope was used to detect cell-DAPI-labeling ratio.At the same time,some cells labeled with DAPI were cultured as before to find out whether the DAPI-labeling had some side effects on the vitality of the labeled BMMNCs.After myocardial infarction was induced by left anterior descending artery ligation,the ischemic area was directly injected with L-DMEM in control group and BMMNCs in group of transplantation of BMMNCs immediately after AMI;1 week later, BMMNCs were transplanted into group of transplantation of BMMNCs 1 week after AMI.Cardiac function and structure were evaluated by echocardiography before the operation and on the day 8 weeks post-infarct.Fluorescence microscope was used to detect cells labeled with DAPI.The number of vessels and the vasculogenesis in the infracted area were examined by histology study.TUNEL method was used to analyse cardiac myocyte apoptosis in the area far away from ischemic area.Results; 1.The DAPI-labeling ratio of rats BMMNCs was 100%,and labeled cells could survive well in vitro.2.Echocardiography eight weeks post-infarct manifested that,EF was (0.299±0.044 VS 0.318±0.042 VS 0.591±0.092)in control group,group of transplantation of BMMNCs immediately after AMI and group of transplantation of BMMNCs 1 week after AMI respectively,there was no difference between control group and group of transplantation of BMMNCs immediately after AMI,P＞0.05;There were significant difference in EF between group of transplantation of BMMNCs 1 week after AMI and the other two groups,P＜0.001.There was no significant difference in ESAWTR between the 3 groups,each P＞0.05.About ESPWTR,there was no significant difference between control group and group of transplantation of BMMNCs immediately after AMI,P＞0.05;and significant difference between group of transplantation of BMMNCs 1 week after AMI and the other two groups,P＜0.05.3.No DAPI labeled cell was found in all slices.4.Local capillary density in infarcted area was much higher in group of transplantation of BMMNCs 1 week after AMI than in control group and group of transplantation of BMMNCs immediately after AMI(2.14±0.62)VS(2.37±0.74) VS(7.08±1.06),each P＜0.01,but there was no significant difference between control group and group of transplantation of BMMNCs immediately after AMI,P＞0.05.5.Apoptosis ratio in nonischemic area in group of transplantation of BMMNCs 1 week after AMI was much lower than the other two groups,each P＜0.01,and no difference between control group and group of transplantation of BMMNCs immediately after AMI,P＞0.05.Conclusions;1.BMMNCs transplantation 1 week after AMI can improve the heart function 8 weeks after MI in rats;BMMNCs transplantation immediately after AMI has little or no effect on the heart function 8 weeks after MI in rats.2.There is little or no relationship between the improvement of heart function and transdifferentiation of transplanted BMMNCs in the rats 8 weeks post-infarction. 3.BMMNCs transplantation 1 week after AMI increases local capillary density in infarcted area and decrease cardiac myocyte apoptosis,these may help to improve the heart function in rats 8 weeks post-infarction.