| The prevalence of diabetes is increasing popular in the world. The difficult wound healing after tooth extraction is an important complication of diabetes mellitus. An important reason for diabetic refractory wound happened is difficult to form new blood vessels. Many datas suggested that endothelial progenitor cells Is one of the key factors in angiogenesis. Endothelial progenitor cells are the precursor cells of the vascular endothelial cells, after that circulating endothelial progenitor cells undergo further proliferation and differentiation homing to the peripheral tissue to repair the damage endothelial. Current experimental studies suggested that basic fibroblast growth factor can obviously improve proliferation, migration and other functions of EPCs. But whether basic fibroblast growth factor also has a role on endothelial progenitor cells of diabetic damage, there is no relative research reports. Therefore, through this experiment the isolation and culture of endothelial progenitor cells in vitro on diabetes, to investigate the effect of different concentrations of bFGF on endothelial progenitor cells of diabetes, which to lay the foundation for endothelial progenitor cell transplantation.PartⅠ:separation, culture, characterization of different tissue-derived endothelial progenitor cellsObjective:To compare the biological characteristics of endothelial progenitor cells derived from spleen and bone marrow in vitro.Methods:Mononuclear cells derived from bone marrow and spleen of Sprague Dawley(SD) rat were isolated by density gradient centrifugation and inoculated on fibronectin-coated culture flask and cultured with complete medium. Morphologies were observed with an inverted microscope. FITC-labeled UIexeuropaeus agglutinin-I (FITC-UEA-I) and DiI-labeled acetylated low-density lipoprotein (DiI-ac-LDL) double staining of differentiating endothelial progenitor cells were identified by Laser confocal microscopy. Endothelial progenitor cells proliferation was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. Migration of endothelial progenitor cells by Transwell chamber assay, adhesion and nitric oxide(NO) secretion ability of endothelial progenitor cells were compared by relevant experimental determination.Results:The mononuclear cells derived from spleen and bone-marrow were isolated by density gradient separation and identified by differentiating into kinds of blood-island like cell clusters and staining FITC-UEA-I and Dii-ac-LDL together observed by fluorescence microscope. With repect to the biological characteristics, such as proliferation, migration, adhesion and NO production, endothelial progenitor cells from the bone marrow is better than those from the spleen.Conclusion:Compared with the spleen, bone marrow is more preferred for the extraction of endothelial progenitor cells in vitro.PartⅡ:Effect of concentration of basic fibroblast growth factor on proliferation, migration and angiogenesis function of diabetes EPCs.Objective:To investigate the effect of concentration of basic fibroblast growth factor on proliferation, migration and aniogenesis function of diabetes EPCsMethods:To set up the type Ⅱ diabetes model in rats induced by streptozocin and high carbonhydrate fat diet. Mononuclear cells derived from bone marrow of the type Ⅱ diabetes rats were isolated by density gradient centrifugation,inoculated on fibronectin-coated culture flask and cultured with complete medium. Morphologies were observed with an inverted microscope. FITC-labeled Ulexeuropaeus agglutinin-Ⅰ (FITC-UEA-Ⅰ) and DiI-labeled acetylated low-density lipoprotein (DiI-ac-LDL) double staining of differentiating endothelial progenitor cells were identified by Laser confocal microscopy. To investigate the effect of the concentration of basic fibroblast growth factor on proliferation, migration and aniogenesis function of diabetes EPCs. Endothelial progenitor cells proliferation, migration of endothelial progenitor cells, and aniogenesis ability were measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, Transwell chamber assay and Matrigel basement membrane matrix, respectively.Results:The mononuclear cells derived from bone-marrow of the type Ⅱ diabetes rats was isolated by density gradient separation and staining FITC-UEA-I and Dii-ac-LDL together observed by fluorescence microscope. On the part of the biological characteristics, such as proliferation, migration and aniogenesis function, EPCs from the type Ⅱ diabetes rats is weaker than from the normol. basic fibroblast growth factor can improve the proliferation and migration of diabetic EPCs.25ng/mL of basic fibroblast growth factor can significantly promote the proliferation of diabete EPCs and 50ng/mL of basic fibroblast growth factor can significantly promote the migration, but diabete EPCs cannot form tubular structures on Matrigel basement membrane matrix.Conclusion:Diabetes mellitus has greatly damaged the proliferation, migration and angiogenesis capacity of EPCs. Basic fibroblast growth factor can improve the proliferation and migration of diabetic EPCs, but had no obvious promoting effect on the angiogenesis ability in vitro. |
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