The Effect And Mechanism Of Basic Fibroblast Growth Factor On The Formation Of Blast-colony-forming Cell Derived From Embryonic Stem Cells

BACKGROUND: Some studies show that basic fibroblast growth factor (bFGF) strongly expresses during the process of embryonic stem cells differentiating into hematopoietic stem cell, yolk sac blooding, and fetal liver hematopoiesis.OBJECTIVE: To study the regulation of bFGF on the blast colony forming cell (BL-CFC) by adding bFGF in the medium of embryoid body generation.METHODS: 1.The third to fifth generation of the primary mouse embryonic fibroblasts were recovered, and then incubated with the DMEM medium containing mitomycin C for 2.5 hours in order to lose the proliferative capacity. Then cells were suspended into single cell by trypsinization and inoculated in the gelatin-coated bottle at the density of 10×10~4/cm~2. After culturing for 24 hours, mouse embryonic stem cells (mESC) of D3 were recovered and place on the feeder layer cells. 2.According to the composition of medium in embryoid body generation, mESCs were divided into four groups: control group: standard medium + VEGF + SCF; 5μg/L bFGF group : standard medium + VEGF + SCF + 5μg/L bFGF;10μg/L bFGF group : standard medium + VEGF + SCF + 10μg/L bFGF;15μg/L bFGF group : standard medium + VEGF + SCF + 15μg/L bFGF. Each group was cultured for 3 days and 6 days respectively, and the cloning number of BL-CFC was quantified as well as Flk-1+ expression was observed the immunofluorescence.in order to relect the best concentration of bFGF. 3. Analyze some relevant expression of BL-CFC for 3,4,5,6 days respectively.4.To explore the machnisns of bFGF by detecting the expression of relevant receptor and specific t o arget gene f signal pathways.RESULTS: 1.Peparating MEF successfully ;2. Amplificating and frozing a large number of embryonic stem cells ;3.Adding bFGF in the course of embryoid body growth could significantly increased the number of BL-CFC (P < 0.01), and the positive results of Flk-1 and the average absorbance were also increased significantly (P < 0.01). bFGF effectively promoted embryoid body amplification and proliferation of BL-CFC and the best convention is 10μg/L.3.During generation process of the BL-CFC, Flk-1 gradually decreased, while CD133, CD34 expression increased;4. I During generation process of the BL-CFC, Wnt, Shh signaling surface receptor expression increased, their respective specific target gene (Axin2) also gradually increased.CONCLUSION : 1. Peparating MEF and amplificating embryonic stem cells successfully;2. bFGF can promote the formation of BL-CFC and the best convention is 10μg/L.3.BL-CFC can differentiate to the hematopoietic stem / progenitor cells .4 The mechanism of bFGF is to activate Wnt signaling pathway.