| ObjectiveFreshly isolated from human umbilical cord blood progenitor cells, and its culture and identification. Density gradient centrifugation using parcoll isolated from umbilical cord blood progenitor cells (EPCs), the cells will be isolated and purified, and use of cord blood were cultured in vitro, using flow cytometry and factor-related antigen staining of the cultured The cells were identified. The results show that growth can be observed in good condition EPCs. Human umbilical cord blood can be isolated, cultured endothelial progenitor cells, endothelial progenitor cells in favor of further study and clinical application.MethodsObtained by density gradient centrifugation of cord blood mononuclear cells, and then were treated with M199 medium containing fetal bovine serum and autologous serum containing M199 culture medium in vitro, differentiation, and amplification was observed in adherent cells induced morphological changes of differentiation , by immunohistochemistry, immunofluorescence and flow cytometry techniques from different angles on the adherent cells were identified.Results1.1 to 2 days in culture can be seen adherent cord blood mononuclear cells; spindle-shaped cells usually appear after 3 days, after adherent cells gradually increased, and gradually became spindle; when the cells cultured for one week, formed around the spindle cells, the central round cells mainly typical colony; 10d, the spindle cells were observed both linked to the typical line-like arrangement of structures; cell culture to one month, the interconnected cell clusters began to form network-like connections structure. With the increase of culture time, gradually reduce the length of spindle cells and showed a typical cobblestone-like changes.2.Adherent cells were cultured to observe the week, covered with fibronectin in the cell culture medium were significantly higher than not covered with multi-cell fibronectin (t = 8.25, P <0.01).4.Immunohistochemistry showed: CD31, vWF antigen immunohistoche in newly isolated mononuclear cells were negative; mononuclear cells cultured for 2 weeks, FCS-M199 adherent cells cultured CD31 positive expression rate of: (88.62±3.74 )%, vWF expression was: (73.36±3.88)%. AS-M199 adherent cells cultured CD31 positive expression rate of: (86.81±4.26)%, vWF expression was: (73.14±3.54)%.4.Immunofluorescence staining showed that: cell culture to 7 days, FCS-M199 culture of adherent cells staining positive rate (85.04±3.56)%, and DiI-acLDL labeled red fluorescent EPCs can continue in vitro than 6W; AS -M199 culture of adherent cells staining positive rate (82.33±4.57)%.5 . Flow cytometry analysis showed: Week 1 cell surface marker determination adherent CD34, CD133, and KDR were: 25.37%±5.52%%, 45.78%±6.39%, 77.75%±7.50%; 2 weeks of flow cytometry analysis showed that CD133-positive rate of 0, CD34 and VEGFR2 expression rates were 55.13%±7.54%, 84.57%±5.14%.Conclusions1. By density gradient centrifugation from cord blood mononuclear cells, and then VEGF, bFGF, BPE and other factors induced culture to VEGFR-2, CD133 and CD34 as a marker of endothelial progenitor cells is to obtain a simple method.2. Added FN can promote the growth of the adherent EPC.3. AS-M199 medium can be replaced by FCS-M199 medium containing EPCs.4. With DiI-acLDL labeled red fluorescent EPCs expressed lasted for 6 weeks, and this method is simple, feasible, not easy to be contaminated, so, DiI-acLDL EPCs in vivo transplantation ideal tracer.5. Mononuclear cells was found in the past is not easy adherence, this study found that by adding fibronectin to promote endothelial cell adhesion, fibronectin added density was significantly greater than cells without added fibronectin group, cell density (t = 7.45, P <0.01), there are significant differences. |
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