The Effect Of Wnt、 FGF1on Embryonic Stem Cells In Hematopoietic Differentiation

Objective:This experiment use overexpression of Wnt and FGF1in embryoid body to detect the expression of Flk-1+、CD133+、CD34+and explore the expression of Shh, and discuss the influence of Wnt and FGF1in hematopoietic differentiation in mouse embryonic stem cell.Method:1.Culture mouse embryonic stem cells:The purchase of ICR mice, Take the pregnancy to13.5days of mouse used to separate MEF;Use3-5generations of logarithmic growth phase MEF to made feeder layer cells; Embryonic stem cell D3cells were seeded on feeder layer cells,use differential adhesion method to remove feeder cells when they were significantly colony then hanging drops were prepared embryoid bodies collected after48h2.The role of overexpression of wnt in hematopoietic differentiation of mouse embryonic stem cells:Divided into three groups according to the different concentrations of lithium chloride:control group,5mmol/L and10mmol/L of LiCl group, then respective cultured for3,5,7,9days, each proposed embryo Flk-1+CD133+、CD34+cells were measured and analyzed using IMAGE-PRO PLUS image analysis system; Using flow cytometry to detect Flk-1and CD133co-expression in embryoid bodies; The optimal concentration of5mmol/L group by RT-PCR detection of Flk-1gene expression.3.The expression of Shh during overexpression of Wnt:To detect the expression of Shh in embryoid bodies by use of immunofluorescence, analyzed using IMAGE-PRO PLUS image analysis system.To explore the machnisns of Wnt by detecting the expression of Shh in embryoid bodies.4.The role of FGF1in hematopoietic differentiation of mouse embryonic stem cells:To detect the expression of FGF1in embryonic stem cells and embryoid bodies by use of immunofluorescence,The hanging drop method to collect48h embryoid bodies then suspension to continue training, add different concentrations of FGF1to the differentiation medium and then respective cultured for3,5,7,9days to collection of embryoid bodies, use flow cytometry to detect Flk-1and CD133positive cells rate in embryoid bodies.Results:1.Successfully prepared a large number of mouse embryonic fibroblast、feeder layer cells、embryonic stem cells;2.(1)Overexpression of wnt signaling,Flk-1and CD133-positive cells are different at different times with different concentrations. the results show that cultured5d group was significantly higher than other groups(P<0.05) in different times of Flk-1positive cells in the mean absorbance value;5mmol/L groupwas higher than other groups(P<0.05) in different concentrations of Flk-1positive cells;and CD133positive cells results show that cultured5d group and control group were higher than other groups(P<0.05) at different times and concentrations; Experiment groups have a higher CD34+cells compared with control groups(P<0.05),5mmol/L group was significantly higher than other groups(P<0.05) in the same times but first growth and then decline in the same concentrations;(2) The flow cytometry results showed that the co-expression positive cell rate was highest in the5d of5mmol/L group,P<0.05;(3) RT-PCR detect5mmol/L group at different times of Flk-1expression and find5d was the most obvious.3. Shh positive cells results show that5mmol/L and5d groups were higher than other groups in different concentrations with different times(P<0.05).4. FGF1have the expression in embryonic stem cell and embryoid bodies;After different concentrations of FGF1treatment, we found Flk-1+and CD133+cells was lower than control groups in5days but increased after7day, P<0.05.Conclusion:l.Amplified and frozen a large number of mouse embryonic fibroblast、feeder layer cells and mouse embryonic stem cells;2.Wnt can promote the formation of Flk-1+、CD133+、CD34+cells and increase the expression of Shh in the embryoid body, but the function by impact of time and concentration, the optimum concentration of5mmol/L and the best time was on5d; 3. The signaling pathway of wnt can regulate the expression of shh in a concentration-time-dependent characteristic, we can explore that the machnisns of Wnt on embryonic stem cell in hematopoietic differentiation related to Shh.4. Initial proof that wnt and FGFlplay an important role in mouse embryonic stem cells of hematopoietic differentiation.