The Experimental Study Of Different Ways Of Transplanting Bone Marrow Mesenchyma Stem Cells Into Rats With Myocardial Infarction

Objective:In order to provide further experimental evidence and clinic feasibility for treating acute myocardial infarctions (AMI) through the transplanted bone marrow-derived mesenchymal stem cells (BMMSCs),we compared the effects and impacts of myocardial infarct area,which treated SD rats with acute myocardial infarctions through myocardial and intravenous injected BMMSCs respectively.Methods:1. Culture and label of BMMSCs in rats:took two male SD rats with body weight 50-100g, collecting their bilateral femur and tibia bone marrow under sterile conditions, cultured primary BMMSCs using bone marrow adherent separation and density gradient centrifugation, cells were amplified and purified through timely and repeated passage, labelled as the third generation with fluorescent dyes CM-DiI in vitro to be BMMSCs source of donated cells.2. Establishment of SD rats model of myocardial infarction and transplantation of BMMSCs:the 40 female SD rats of Specific Pathogen Free(SPF) level of 200-250g weight were anaesthetized with 10% chloral hydrate intraperitoneal. After linking with respiration machine, their left anterior descending branch (LAD)were ligated.Then the myocardial infarction model of SD rats had been builded.Fourty rats were randomly divided into four groups:Group A (myocardial injection group,n=10):were transplanted 5x106 BMMSCs which were labeled CM-DiI after myocardial infarction model had been builded for one hour; Group B(intravenous injection,n=10):were transplanted 5x106 BMMSCs which were labeled CM-DiI by tail injected after myocardial infarction model had been builded for one week; Group C(control group, n=10):were transplanted in the tail vein myocardial infarction in non-transplant serum-free modified BMMSCs dulbecco's modified eagle's medium(DMEM) after one week, about 0.5ml; Group D(sham-operation group,n=10):through the heart with a needle thread ligation in rats, but not LAD, underwent myocardial biopsy around the HE staining four weeks after that.3. Comparing the changes of myocardial infarct size by using dual source computed tomography to check and 2,3,5-triphenyl tetrazolium chloride (TTC) staining.To measure infarct size by using dual source CT and TTC in rats, used SPSS to analyse datas, and finally marked survival with CM-Dil fluorescence microscope BMMSCs after four weeks.Results:1. BMMSCs adherent within 12-24h after inoculation, and most of hematopoietic stem cells and red blood cells in suspension, suspension cells can be removed by changing the medium several times. After growing 5-6 days, increased volume of adherent spindle-shaped cells, some cells were polygonal, around cells were paved in single layer in 7-12 days, arranged in spiral-shaped, in fusiform or polygonal. BMMSCs rats primary cell cultures can be subcultured in7-12 days. The third generation BMMSCs was labelled with CM-Dil in vitro, the mark rate of labelled BMMSCs was up to 95% under fluorescent microscopy.2. The myocardial infarction model of SD rats by the ligation of LAD was successfully established. HE staining showed myocardial was mortified and scar tissue was formed in myocardial infarction area.3. Dual source CT and TTC staining had good correlation, r=0.975; SD rats were successfully transplanted BMMSCs and survived in A, B, C, D four groups:Group A reduced less infarct size significantly compared to Group B, P value of 0.001; Group A decreased less infarct size obviously compared to Group C, P value of 0.001,; Group B reduced less infarct size compared to Group C, P value of 0.001; seen CM-Dil labeled nuclei of the frozen myocardial infarction tissue sections were showed red fluorescence of two ways to transplant,it may indicate that transplanted cells survived in the host myocardium.Conclusion:1.BMMSCs can be cultured with bone marrow adherent separation and density gradient centrifugation; Bone marrow mesenchymal stem cells can be labelled by CM-DiI.The labeled effect is good, and worth.to be promoted.2. Coronary artery ligation model of myocardial infarction is the reliable and stable model of myocardial infarction.3. Mesenchymal stem cells can be returned to myocardial infarction after intravenous transplanting, the myocardial infarct size in transplanted rats was significantly smaller than that after intravenous transplantation, transplanted cells was able to survive in the host myocardium.