| Research Background:Acute myocardial infarction (AMI) is a common disease that severly threatens the elder's health. At the present time, the ultimate treatment to regenerate the infarcted myocardium and the pathological coronary arteries is lacking.Although dissolving the thrombus and catheterizing in time in 4-6 hours after infarction breaks out can restore the blood of the infarction-associated vessels and save the dying cardiomyocytes, the reperfusion therapy can only limit the infarcted size which has no effect on the infarcted myocardium, and the ischemic cardiomyocytes of the majority patients have infarcted when they go to hospital. Lacking the abilty of differentiation , the infarcted myocardium can only be replaced by the fibroblasts to form scar tissue and has no systolic function. Thus the patients are easy to suffer from heart failure, even sudden 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 candidates in this field in the present time. It is indicated recently that mesenchymal stem cells(MSCs) are multipotent tissue stem cells that can be induced in vitro to differentiate inot osteroblasts, chondroblasts, tendon, ligament, fibroblasts, nerve cells and muscles, et al. In addition, MSCs are easy to be separated and expanded in vitra, and they don't express the costimulation molecule B7, et al. All these characters make MSCs the focus of cardiology and may become the ideal candidates to regenerate cardionmyocytes after myocardial infarction. Objectives:(1) To establish an effective method for isolation, purification and cultivation of human MSCs; (2) To compare the characteristics of human MSCs from adult bone marrow and from fetal livers, including their phenotype, multipotential and immunological properties; (3) To investigate the possibility to make human MSCs differentiate along cardiac lineage and to find the way to get high differentiation rate in vitro; (4)To investigate the survival and cardiac differentiation of human MSCs when transplanted into infarcted hearts of rats, which were at different immunological states. At the same time, to investigate the immunological reaction of rats after being transplanted with human MSCs. Methods and results: Part â… :We examined the isolation,purification, expansion and multi-differentiation of human MSCs from adult bone marrow (AMSCs) and fetal livers( FMSCs). Mononuclear cells ,isolated from bone marrow or fetal livers, were isolated with 1.073g/mL Percoll. MSCs were obtained by removing the non-adherent cells and were cultured in DMEM-LG medium with 10% fetal calf serum(FCS). The MSCs were purified and expanded through passaging in time. Each primary culture was replated to 2-3 new plates when cell density within colonies became about 80% confluent approximately 5-6 days after replating.After each passage, the number of AMSCs increased about 2.6 doubles, while FMSCs about 12.5 doubles.The cells kept their fibroblast-like morphology and their proliferation rate until passage 20. Cells from passage 2 to 9 were used for the following test. TheFACS results showed that both AMSCs and FMSCs expressed antigen CD29, CD44, CD166, SH2(CD105),SH3 and SH4, and didn't express CD14,CD34 and CD45. MSCs were induced to differentiate along osteogenic, chondrogenic and adipogenic lineages under appropriate stimulus, which was indicated by calcium depositin seen with the von Kossa method, positive staining for glucosaminoglycans with alcian green and positive staining for neutral lipid vacuoles with oil red O. The results indicate: 1. We got a large quantity of purified AMSCs and FMSCs, whose properties were stable; 2. FMSCs have stronger expansion potential than AMSCs in vitro, so a huge number of FMSCs could be obtained within short period; 3. Both FMSCs and AMSCs have multipotentials to be induced to differentiate along osteogenic, chondrogenic and adipogenic l...
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