In Vitro On Biological Character Of Embryonic Hepatic Stem Cells

Research Background: Coronary atherosclerotic heart disease,Acute myocardial infarction (AMI) included, is a common disease that threatens the human being's health severely. The ultimate treatment to generate the infracted myocardium and the pathological coronary arteries is lacking at the present time. Although dissolving the thrombus and catheterizing 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 enough ability of differentiation, the infarcted myocardium can only be replaced by the fibroblasts to form scar tissue and has no systolic function. How to treat AMI more effectively is the focus in the medical field. The cellular transplantation technique gives us a good prevision to regenerate the injured myocardium.Fetal cardiomyocytes, embryonic stem cells and mesenchymal stem cells became the candidates in the field in the present time. Fetal cardiomyocytes and embryonic stem cells face the ethic limitation and the immune reaction. More and more studies base on mesenchymal stem cells(MSCs) for outstanding biological characters and bone marrow stem cells(BMSCs) and embryonic hepatic stem cells(EHSCs) is the emphasis especially.It is indicated that bone marrow stem cells(BMSCs) are multipotent tissue stem cells that can be induced in vitro to differentiate into tendon, ligament, fibroblasts and muscles, et al. In addition, BMSCs don't express the costimulation molecule B7, et al. All these characters make BMSCs the focus of cardiology and may become the ideal candidates to generate cardiomyocytes after myocardial infarction. But evidence shows that EHSCs have the more capacity to proliferate and the less immunity than BMSCs, so EHSCs are focused on more and more.Objective:(1)To improve the method for isolation, purification and cultivation of mouse EHSCs; (2)To compare the characteristics of mouse EHSCs in different embryo duration; (3)To investigate the possibility to make mouse EHSCs differentiate along cardiac lineage; (4)To compare the differentiation states of EHSCs which are in different conditions.Methods and results:Part I: We examined the isolation, purification, expansion and compared the characteristics of mouse embryonic hepatic stem cells in different embryo duration ( ED 13.5 day , ED 16.5 day and ED 19.5 day). Collagenase and EDTA digestion and the method of adhering to culture plastic in different time were used to isolate EHSCs from mouse fetal liver which were then cultivated by L-DMEM containing 15% fetal bovine serum. We got the result that the cells of the least difference in morphology, the best situation of growing and the most evident characteristics of stem cells were obtained from ED13.5 day group and the morphological difference between cells and the ALB and CK19 expression increased yet growing situation decreased gradually with the increasing embryo duration.Conclusion: It is suggested that a majority of primordial progenitor cells exist in early age of fetal liver and differentiate into ALB+CK19+ stem cells gradually and a large quantity of EHSCs whose properties were stable were obtained by the improved methods .Partâ…¡:We investigated the possibility to induce EHSCs to differentiate along cardiac lineage and compared the differentiation states of EHSCs which are in different conditions. Cells from passage 3-4 were planted at the density of 1.5×10⁴ /cm² and were treated with the combination of 5-azacytine(5-aza) and DMSO in different doses for different hours. Cells, treated with 5-aza of 5μmol/L and DMSO of 0.8% for 24 hours, cultured at 37OC,5%CO₂ and 20%O₂, showed the sign of cardiac differentiation and stained positive for troponin T andα-actin 4 weeks after treatment. Conclusion: EHSCs have the potential to differentiate along cardiac lineage; The stimulus for the cardiac differentiation of mice EHSCs is different from other source.Summary for this thesis:1. A large quantity of EHSCs whose properties were stable can be obtained by the improved methods of adhering to culture plastic in different time.2. A majority of primordial progenitor cells exist in ED13.5 day of fetal liver.3. Mice EHSCs can be induced into cardiomyogenic cells by the combination of 5-aza and DMSO.4. The moderate inducer which can induce EHSCs into cardiomyogenic cells is the combination of 5-aza of 5μmol/L and DMSO of 0.8% and higher dose inducer can't improve the induce efficiency.5. The moderate treatment time is 24 hours with inducer includes 5-aza of 5μmol/L and DMSO of 0.8% and longer treatment time can't improve the induce efficiency.