| Bone marrow of adults contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells and have therefore been termed endothelial progenitor cells (EPCs).EPCs emigrating from special tissue home to sites of neovascularization and differentiate into endothelial cells(ECs) in situ.They promote local oxygen perfusion and revascularization process to maintain the function of ischemic tissue through vasculogenesis and angiogenesis.Therapic cells transplation has underdone in ischemic diseases treatment and provide to be effective.Therefore the deficiency of EPCs hinders its clinical application.How to obtain optimized quality and quantity of EPCs through economical method requires further study.AIMEPCs has capacity to take part in neovascularization in ischemic tissue which is useful in clinical treatment,but there is no mature method to isolate andproliferate EPCs effectively. Furthermore, diverse method in EPCs isolation and proliferation used in abroad are much more expensive which will block the investigation in EPCs.Our aim is finding out a method to isolate and proliferate EPCs economically and effectively for its further study in basic theory and clinical treatment.MethodsPart one: The proliferation and differentiation of mononuclear cells derived from bone marrow in endothelial basal medium.Mice bone marrow is obtained under sterile condition,then mononuclear cells were isolated by density gradient centrifugation.The cells are divided into three groups,which is cultured in different media contained diverse ECGS concentration respectively:A groupxontain ECGS 300ug/ml;B groupxontain ECGS 200ug/ml;C groupxontain ECGS 100ug/ml.In different culture point,total cells amount,positive percent in CD34,vWF and eNOS are analysed by FACS.At the culture end,the cells are were stained with FITC-labeled vWF and eNOS.and are evaluated by microscope.Part two: The proliferation and differentiation of bone marrow derived mononuclear cells in diverse endothelial culture conditions.To isolate mononuclear cells following the method described above,then divide cells into three groups,which are plated in diverse culture conditions:A group: endothelial basal and coculture with HUVEC;B group: endothelial basal medium with VEGF and bFGF;C group: endothelial basal medium. In different culture point,total cells amount,positive percent in CD34,vWF and eNOS are analysed by FACS.ResultsPart one:1 .In fresh isolated bone marrow derived mononuclear cells,CD34+ cells accountfor (42.3 +2.3)%,percent of vWF or eNOS positive cells less than 1%.2.BM derived MNCs are plated in basal endothial medium contained diffenentECGS concentration.The total amount of cells is increasing respectivelyaccording to culture days.In general,there is significant statistics differencebetween A ,Bgroup and C group(F=369.177, P=0.000);no statistics difference isfound between A and B group(F=3.3400,P=0.061).3. The percent of vWF positive cells is increasing respectively according to culture days.In general,there is significant statistics difference between A ,Bgroup and C group(F=238.584, P=0.000);no statistics difference is found between A and B group (F=20.1600, P=0.11).4. The percent of eNOS positive cells is increasing respectively according to culture days.In general,there is significant statistics difference among three groups(F=33.904, P=0.001).5. Fluorescence stain in situ:At the culture end,the cells form capillary network,then are were stained with FITC-labeled vWF and eNOS.The cells are evaluated by microscope, which present positive response.Part two1. .In fresh isolated bone marrow derived mononuclear cells,CD34+ cells account for (47.1 + 5.2)%, no vWF positive or eNOS positive cells are found.2. The total amount of cells is increasing respectively according to culture days.Ingeneral,there is significant statistics different among three groups(F=661.3,P=0.000).After 3 days,no significant diffenent among them,but...
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