Effect Of PAX6 On Proliferation And Differentiation Of MESCs And Research On Related Signal Molecules

Objective: To study the effect of PAX6 on the biological characteristics of mouse embryonic stem cells(mESCs)and its potential mechanism.Methods The transfection with over-expression and interference vector of PAX6 was performed on mESCs cells respectively,then the transfected cells were identified by PAX6 and GFP immunofluorescence staining,and verified by Western blot.The effects of PAX6 on the proliferation of mESCs were detected by CCK8 assay.Western blot was used to detect proteins K5,K14,p-PI3 K,PI3K,p-AKT,AKT,p-P70S6 K,P70S6K,GLUT1 expression levels.Flow cytometry was used to detect the effect of PAX6 on cell cycle of mESCs.Morphological changes of mESCs cells on day 7 were observed under the microscope.Results Fluorescent expression of GFP and PAX6 were both observed in mESCs with transfected PAX6 only.Compared with the control group,the expression of PAX6 in PAX6 over-expression group was significantly increased(P<0.05),and the difference was statistically significant.Compared with the control group,at the same time node,PAX6 with interferenced vector could promote the proliferation of mESCs cells,while the over-expression one was opposite.With the extension of time,the effect of interference vector on promoting the proliferation of mESCs was more obvious.Compared with the control group,the expression of K5 and K14 in PAX6 over-expression group increased significantly.The cell cycle block of mESCs in PAX6 over-expression group was observed.The morphological changes of mESCs cells on day 7 were observed under light microscope.The results showed that mESCs in the four groups were round or elliptical with clear boundaries.Compared with the control group,the cells of interference group were loose,while ones of over-expression group were closely arranged and clustered,showing a transition to limbal stem cells.Compared with the control group,the expression of p-PI3 K,p-AKT,p-P70S6 K,and GLUT1 in PAX6 over-expression,PAX6 over-expression & BKM120 group and BKM120 group decreased significantly(P < 0.05),and the content of ATP and glucose in mESCs decreased in varying degrees(P < 0.05)Conclusion PAX6 could suppress the proliferation of mESCs by inhibiting the activity of PI3K/AKT signaling pathway and decreasing the ATP and glucose content of mESCs.PAX6 could induce the differentiation of mESCs into LSCs and promote the expression of marker proteins K5 and K14.