| Endothelial progenitor cells (EPCs) is a group of stem cells specifically homing toangiogenic organization and differentiate into endothelial cells in the body after birth，including cell population of a certain stage from hemangioblast to fully differentiatedendothelial cells, which not only involved in embryonic angiogenesis, but also closely relatedto vascular endothelial injury healing and angiogenesis. Endothelial progenitor cells are theprecursor cells of the vascular endothelial cells, within the bone marrow reside in closecontact with surrounding stromal cells and are released upon activation by physiological orpathological factors, after that circulating endothelial progenitor cells undergo furtherproliferation and differentiation homing to the peripheral tissue to repair the damageendothelial. Recently a number of studies have shown that the EPCs is the main cells involvedin physiological and pathological angiogenesis after birth. Thus, endothelial progenitor cellsplay an important role in cardiovascular and cerebrovascular diseases, and tumor angiogenesisand wound healing and other aspects, has broad application prospects.Endothelial progenitor cells could be applied for clinical treatment for its function,including the repair of injured vessel wall, the neovascularization or regeneration of ischemictissue, and the coating of vascular grafts. The most important reason to limit its clinicalapplication is the EPC number isolated and cultured from bone marrow and peripheral bloodis very limited, can not meet the needs of experimental research and clinical applications. Theway to solve this problem, including culture human endothelial progenitor cells from cordblood or umbilical cord tissue, or use cytokines, growth factors, drugs to promote themobilization of human endothelial progenitor cells.Therefore, the study on the culture methods of human endothelial progenitor cells is stillnecessary. In order to culture and expand the number of steady EPCs, In our study, wereferenced the culture and identification of EPCs to a number of domestic and foreignlaboratory programs, by repeatedly trial in the experiment, to compare several importantfactors in the impact of EPC culture and amplification, to compare the culture of endothelialprogenitor cells from human bone marrow, cord blood, umbilical cord tissue, and respectivelyco-culture with different concentrations of VEGF and EPCs to research the promote effect ofVEGF on mobilization of human endothelial progenitor cells, and then select the trainingprogram to get human endothelial progenitor cells of high number and stability progenitor cell characteristics, better ability of proliferative capacity and the ability of angiogenesis. lay agood foundation for transplantation endothelial progenitor cells for the treatment ofcardiovascular disease and multiple organ dysfunction syndrome.This study is divided into three parts:PartⅠ: Isolate and culture different tissue-derived endothelial progenitor cellsObjective:By compare the culture of endothelial progenitor cells from human bonemarrow, cord blood, umbilical cord tissue to establish the standardized separation and cultureand amplification methods of human EPC, to lay the foundation for endothelial progenitorcell transplantation.Methods: Density gradient differential adherent culture and organizations explantmethod was used to culture and isolate endothelial progenitor cells from bone marrow,umbilical cord blood and umbilical cord tissue. According to different seeding density,medium change time, basic culture medium, serum concentrations,divided into severalgroups, to compare the primary EPC colony number and EPC number, to compare the cellgrowth curve and proliferation times of matured EPC, using SPSS statistical software tosummarize the relevant data, analysis, comparison.Results: We successfully isolated, cultured and amplified endothelial progenitor cellsfrom the bone marrow, umbilical cord blood and umbilical cord tissue, the cell. morphologyof EPCs cultured by these conditions and methods are basicly similar. But the cellmorphology will change if bone marrow-derived endothelial progenitor cells be passagedmore than3generations, the endothelial progenitor cells derived from umbilical cord bloodand umbilical cord tissue can be stablely passaged more than10generations with nosignificant change in cell morphology. Human endothelial progenitor cells from umbilicalcord tissue and umbilical cord blood have more stable morphological characteristics ofprogenitor cells and stronger proliferative capacity compared with bone marrow-derivedhuman endothelial progenitor cells.Conclusion:Human endothelial progenitor cells from umbilical cord tissue and umbilical cord bloodhave more stable morphological characteristics of progenitor cells and stronger proliferativecapacity compared with bone marrow-derived human endothelial progenitor cells. The culturemethod of umbilical cord tissue-derived human endothelial progenitor cells can get highernumber and the culture method is simple and reliable, can be applied to the final clinical use for prevention and treatment of multiple organ dysfunction.Part Ⅱ: Identification and function tests of endothelial progenitor cellObjective:Identification of cultured human endothelial progenitor cells and amplified to determineits function, to ensure the quality and purity of the separated and cultured cells.Methods:Through the use of cell morphology and cell ultrastructure to observe the progenitor cellscharacteristics， the use of two-color fluorescence staining from uptake Dil complexdacetylated low-density lipoprotein (Dil-Ac-LDL) and FITC conjuated Ulex europaeus lectin-1(FITC-UEA-1) to observe the phagocytic function，the use of flow cytometry (FACS) andimmunohistochemical expression of CD133, CD34and CD31, KDR phenotype toidentification EPC, the use of angiogenesis in vitro test to identification EPC.And to comparethe function of the different tissue-derived endothelial progenitor cells.Results:Cell morphology and cell ultrastructure of the endothelial progenitor cells are in line withthe endothelial progenitor cell characteristics, double staining rate by Dil-ac-LDL andFITC-UEA-1can reached over80%，The positive rate of CD133、CD34、CD31、KDR wereaccordant with in line with the EPC phenotypic characteristics by immunohistochemicalmethod and flow cytometry detection，successful in vitro vascular capacity.Conclusion:In this study, the results from a comprehensive identification system(morphologicalfeatures, immunohistochemistry, flow cytometry, two-color fluorescencestaining,angiogenesis in vitro test) fully show that the cells we cultured are humanendothelial progenitor cells. Human endothelial progenitor cells from umbilical cord tissueand umbilical cord blood have more stable progenitor cell characteristics and stronger into theblood vessels ability compared with bone marrow-derived human endothelial progenitor cells.Part Ⅲ: The mechanism of how VEGF promote the proliferation of the people umbilicalcord tissue-derived endothelial progenitor cell.Objective:To research the role of VEGF to promote the proliferation of human umbilical cordtissue-derived endothelial progenitor cells. Methods:Comparison of number changes and cell culture fluid nitric oxide (NO) levels in differentconcentrations of VEGF co-cultured with human umbilical cord tissue-derived endothelialprogenitor cells. Deal the umbilical cord EPCs with PI3K specific inhibitor LY294002, at thesame time set the blank group and solvent (DMSO) control group, and10ng/mlVEGF+inhibitor LY294002group, then detection the expression of phosphorylated Akt protein bywestern blot, for the determination of cell culturesolution of nitric oxide (NO) levels bynitrate reductase method, detected the cell proliferation and angiogenic capacity after addinhibitors.Results:Concentrations lower than10ng/ml of VEGF co-cultured with human umbilical cordtissue-derived endothelial progenitor cells, the number of endothelial progenitor cells andnitric oxide (NO) levels of cell culture fluid were significantly increased. But higherconcentrations than20ng/ml of VEGF co-cultured with human umbilical cord tissue-derivedendothelial progenitor cells, the number of endothelial progenitor cells and cell culture fluidnitric oxide (NO) levels did not change significantly compared with low concentrations ofVEGF. Add in specific inhibitor of PI3K, LY294002inhibition of PI3K, the expression ofphosphorylated Akt protein was significantly reduced，nitric oxide (NO) levels of cell culturefluid was significantly reduced，cell proliferation and in vascular capacity was significantlydecreased.Conclusion:Concentrations lower than10ng/ml of VEGF co-cultured with human umbilical cordtissue-derived endothelial progenitor cells can significantly promote the proliferation ofhuman endothelial progenitor cells and nitric oxide (NO) levels of cell culture fluid,. Higherconcentrations than20ng/ml of VEGF co-cultured with human umbilical cord tissue-derivedendothelial progenitor cells can not obviously promote proliferation of human endothelialprogenitor cells compared with lower concentrations of VEGF maybe because the receptorsaturation. Add in specific inhibitor of PI3K, LY294002inhibition of PI3K, the expression ofphosphorylated Akt protein was significantly reduced，nitric oxide (NO) levels of cell culturefluid was significantly reduced，cell proliferation and angiogenic capacity was significantlydecreased， suggesting that the VEGF promote the proliferation of human endothelialprogenitor cells by activate PI3K/Akt/eNOS pathway.In short, by compared the culture of endothelial progenitor cells from human bone marrow, cord blood, umbilical cord tissue,we discover that human endothelial progenitor cellsfrom the umbilical cord tissue and cord blood have more stable morphological characteristicsof progenitor cells and progenitor cell characteristics, and stronger proliferative capacity andinto the blood vessels ability than bone marrow-derived endothelial progenitor cells. Theculture method of umbilical cord tissue-derived human endothelial progenitor cells can gethigher number and the culture method is simple and reliable. By research the role of VEGF topromote the mobilization and proliferation of human umbilical cord tissue-derived endothelialprogenitor cells, we discover that concentrations lower than10ng/ml of VEGF co-culturedwith human umbilical cord tissue-derived endothelial progenitor cells can significantlypromote the proliferation of human endothelial progenitor cells and nitric oxide (NO) levelsof cell culture fluid, suggesting that the VEGF promote the proliferation of human endothelialprogenitor cells by activate PI3K/Akt/eNOS pathway. But higher concentrations than20ng/mlof VEGF co-cultured with human umbilical cord tissue-derived endothelial progenitor cellscan not obviously promote proliferation of human endothelial progenitor cells compared withlower concentrations of VEGF maybe because the receptor saturation. This conclusion lay agood cytological foundation for transplantation of endothelial progenitor cells to preventionand treatment of multiple organ dysfunction syndrome in our further research. |
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