Effect Of Sp5 Gene Overexpression In Mouse Palatal Embryonic Mesenchymal Cells On The Expression Of Wnt Pathway Factors Such As GSK-3β

Objective:To investigate the effects of mouse embryonic palatal mesenchymal cells(mEPMCs)overexpression of Sp5 gene on Wnt signaling pathway related factors and cell proliferation,and to explore whether Sp5 gene is involved in palatal development by regulating Wnt signaling pathway.Methods:The bilateral palatal processes of ED14.5 fetal rats were extracted,and primary mEPMCs were obtained by trypsin digestion method,and identified by immunocytochemistry(ICC);The optimal multiplicity of infection(multiplicity of infection)was determined by gradient experiment Methods,mEPMCs were divided into two groups: Sp5 overexpression group(experimental group)and Sp5 empty lentivirus group(control group),the overexpression of Sp5 gene was detected by RT-q PCR;The effects of Sp5 overexpression on the expression of Wnt3 a,GSK-3β and β-catenin in mEPMCs were detected by RT-q PCR and Western blot;CCK-8 method was used to detect the effect of Sp5 gene overexpression on the proliferation of mEPMCs.Results:(1)ICC staining showed that vimentin(Vim)was positive and Pan-Cytokeratin(p-ck)was negative in cultured cells,which was consistent with the characteristics of mesenchymal cells;(2)When MOI = 30 and Polybrene = 4μg/ml,the transfection effect was the best.RT-q PCR detection showed that the m RNA expression of Sp5 gene in the experimental group was significantly higher than that in the control group(P < 0.05),indicating the successful overexpression of Sp5 gene in mEPMCs;(3)RT-q PCR and WB results showed that the expression of GSK-3β,β-catenin and Wnt3 a in the experimental group was lower than that in the control group(P < 0.05);(4)CCK-8 results showed that Sp5 gene overexpression in the experimental group inhibited the proliferation of mEPMCs.Conclusion:(1)Overexpression of Sp5 gene in mEPMCs can reduce the expression of Wnt signaling pathway related factors GSK-3β,β-catenin and Wnt3a;(2)Sp5 gene may be involved in regulating the proliferation of mEPMCs through Wnt signaling pathway.It is concluded that Sp5 gene may participate in palatal development by regulating Wnt signaling pathway.