| Over decades, the research on stem cells to treat degenerative diseases and necrotizing diseases, namely cell replacement therapy, has been a hot topic. Among the large amount of cell types in cell therapy of coronary heart disease like myocardial infarction or heart failure, endothelial stem/progenitor cells is a promising source of neovascularization. Since homing and migration mechanisms are complex, homing and cell regeneration of EPCs has not been unable to have clinical application. In this study, we resort to basic research as a means to explore new hEPCs homing regulatory proteins (namely Rac protein) and possible signaling pathways.This study was to validate the important role of the Rac protein in EPCs homing, thus to provide a new pathway of cell therapy for coronary heart disease. The experiment is divided into the following three parts:Part ⅠCulture and identification of Human endothelial progenitor cells and rat endothelial progenitor cells culture.[Abstract] Objective:To establish a laboratory method of culturing human endothelial progenitor cells (hEPCs, unless otherwise noted, all are referred to isolated hEPCs in our experiments) to explore the culture conditions, cell growth state, and cellular form. Cell culture conditions and growth state are compared with that of the rat EPCs. Methods:The human umbilical cord blood of80-120ml/bag, firstly wet assorted the mononuclear cells using magnetic bead assisted cell sorting assay, after sub-elect of CD133+/VEGFR2cells for flow cytometry, we found that double positive cells accounted for48.79%. These cells were differentially adherent cultured for1week and sent to take conventional photography, immunofluorescence staining photography and cell polarity research. Results (1) hEPCs resemble cable strip or oval shape in the light microscope.(2) The sorted cells have phagocytosis of the DII-acLDL, FITC-UEA dye under fluorescence microscopy and specific color to prove that the cells have a phagocytic function, inferred to be EPCs.(3) After the use of MACS screening, CD133+/VEGFR2+cells accounted for48.79%of all cells.(4) The growth activity of rEPCs isolated from the bone marrow was significantly superior to that of hEPCs. Conclusions (1) The culturing method can yield actively surviving hEPCs, which have cable strip, oval shape in the light microscope and high concentration. hEPCs can be used in the future experiments; but they have worse growth state than that of rat EPCs;(2) differential wall-attachment cultured hEPCs are cultured for5days, and can swallow the DII-acLDL, UEA dye with specific color. Part ⅡSDF-1α’s effect on human endothelial progenitor cell polarity and migration[Abstract] Objective:changes of isolated EPCs’ polarity and cytoskeletal proteins, receptors distribution, cell migration under fluorescence microscopy induced by SDF-1. We used the action board Zigmond chamber to observe the migration of EPCs.Methods:We observed EPCs’ morphology induced by different concentrations of SDF-1α at different time points under light microscopy. FITC and overall Rac fluorescence protein dye were used and three indicators were observed:CD133, GBP/Rac, the F-actin (actin fibers or actin). Results:(1) light microscopy showed that the cell became elongated, stretching of their cell body, the poles of the "polarization" phenomenon.(2) changes in the distribution of cell surface receptors, the role of SDF-1α group (25ng/ml,50ng/ml,100ng/ml,200ng/ml) at1h,2h,6h,12h,24h. CD133, GBP/Rac F-actin expression was the SDF-1dose dependent (at least compared with the blank control group), ie, a positive correlation with the concentration of SDF-1; in SDF-1α200ng/ml dose group and CD133, GBP/Rac, F-actin expression was highest at the12hours time-point, rather than24hours. CD34expression was time dependent, the highest expression at24h, the expression of Racl is not time dependent, the highest expression at12h. Among cell surface receptors of CXCR4, CD18, Integrina4, the expression of CXCR4was time-dependent with the highest expression at24hours, the expression of CD18and Integrina4was not time dependent.(3)We observed the migration of EPCs (24hours) in the Zigmond chamber chemotaxis analysis dish. Through the establishment of the overall EPCs migration vector (vertical Y and horizontal X) analysis, we can calculate the angle and degree of EPCs migration, and have quantitative proof of the strengthening of the directional migration ability of SDF-1under the action of EPCs. Conclusions: EPCs have SDF-1induced polarity changes, regardless of changes in cell morphology or the distribution of the cell receptor, cell populations of EPCs have directional migration to SDF-1α. Part ⅢPrimary research on Rac proteins’molecular mechanism regulated by SDF-1αAbstract Objective:to find out dose of SDF-1and the time-point of the highest expression of Racl, Rac2in SDF-1α induced EPCs using PCR assay combined with the second part of the receptor distribution in the SDF-1concentrations and changes over time. To explore the in vitro dose of SDF-1and the time-point to induce the strongest "homing ability hEPCs,. Whether the expression of CXCR4and p38MAPK related to observation of SDF-1induced Rac. Methods:We divided the co-culture time of hEPCs with SDF-1to5different intervention time points (1h,2h,6h,12h,24h) with SDF-1co-cultured with hEPCs, we extracted RNA from the cells of Rac to measure the mRNA expression changes in the simultaneous determination of the Rac protein and Rac-GTP protein expression changes. We respectively use the CXCR4inhibitor AMD3100and p38MAPK blocker SB203580to intervene SDF-1and hEPCs’ culture, and then measure the protein expression of Rac protein and of Rac-GTP.Results:PCR electrophoresis pattern observed in racl and rac2has the highest expression at12hours. We explored the SDF-1α induced expression of Rac protein and the protein of Rac-GTP in EPCs, according to western OD value, the highest expression at12hours. Rac and its activation pattern of Rac. Joining the CXCR4 inhibitor AMD3100, racl and rac2protein expression inhibition rates were:57.1%and62.2%. Joining the p38MAPK inhibitor, SB203580, significantly inhibited the SDF-1α-induced Rac-1and Rac-2protein expression. Conclusions:(1) Rac has the highest expression with a SDF-1concentration of200ng/ml, the best time point is at12hours;(2) the SDF-1α induced regulation of Rac expression is CXCR4and p38MAPK dependent. Conelusions1. human EPCs make the SDF-lainduced (divided into1h,2h,6h,12h,24h five time points) polarity changes, both in cell morphology and cell recaeptors change. EPCs cell populations have directional migration towards SDF-1α.2. Co-cultured with200ng/ml SDF-1, the expression of both mRNA and protein of Rac proteins in subtypes of racl and rac2are the highest at12hours. After joining the CXCR4inhibitor AMD3100, Racl and Rac2protein expression was significantly reduced; after joining p38MAPK’s inhibitor SB203580, the expression of Rac-1and Rac-2was also significantly reduced, indicating that the SDF-la increase the expression of Rac protein through CXCR4and p38MAPK signaling pathway.The potential application and novelty of this project1.Having established in vitro cell culture system of hEPCs;2.Having observed hEPCs’directional movement to SDF-1α in Zigmond chamber;3.Having set best stimulating condition of SDF-1to the regulation of Rac expression:SDF-1concentration of200ng/ml, the best time point is12hours;4.Rac expression induced by SDF-1α is both CXCR4and p38MAPK signaling pathway dependent. |
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