Identification Of The Effect Of A Novel Wnt Surrogate Protein That Activates The Wnt Signaling In Brain Cells

Objective The purpose of this study is to investigate the effect of a novel Wnt surrogate protein(Wnt-S),on the Wnt/?-catenin signaling pathway in various cells in the brain,and to develop a new strategy for the possible treatment of stroke.Methods Synthesis of an Wnt-S fragment,the method of using molecular cloning will connect this fragment with carrier pc DNA3.1(+),then with E.coli for transformation,plate cloning,pick monoclonal out,bacteria extracted the plasmid,using PEI for HEK 293 F cells transient transfection with the plasmid,take Wnt-S supernatant and purification,Western blot testing Wnt-S containing 6x His-tag,and validation activity in HEK 293 cells.Cultivate HEK 293 cells,human brain microvascular endothelial cells(h CMEC/D3),human brain vascular pericytes(HBVP),human astrocytes(HA),oligodendrocytes(HOC),neural stem cells(NSC)in mice,mice microglia(BV2),rat neurons(Neuron),using immunofluorescence staining method to detect these cells respectively specific markers,Quantitative real-time PCR was used to detect the expressions of receptors FZD1-10(Frizzled family),LRP5/6 and corresponding markers in these cells.These cells were treated with Wnt-S protein,and the expression of Axin2,a target gene of the Wnt/?-catenin signaling pathway,was detected by Quantitative real-time PCR.Wnt-S adenovirus was injected into SGZ region of mouse brain to detect the proliferation of neural stem cells.Results 1.Wnt-S protein can activate the Wnt/?-catenin signaling pathway in HEK 293 cells,and the expression of Axin2,the target gene of the Wnt/?-catenin signaling pathway,increases with the increase of the concentration of Wnt-S protein.2.In human brain microvascular endothelial cells(h CMEC/D3),the Wnt/?-catenin signaling pathway was not significantly activated at low concentrations of Wnt-S protein(?1000ng/ml),but at high concentrations of Wnt-S protein(?1500ng ng/ml).Axin2 expression gradually increased with the increase of Wnt-S protein concentration.3.In human brain vascular pericytes(HBVP),human astrocytes(HA),human oligodendrocytes(HOC),mouse neural stem cells(NSC),and rat neurons(Neuron),Axin2 increases first and then decreases with the increase in the concentration of Wnt-S protein.4.Wnt-S protein could not significantly up-regulate Axin2 expression in mouse microglial cells(BV2).5.In mouse brain,Wnt-S can significantly promote the proliferation of neural stem cells.Conclusions 1.The purified Wnt-S protein has biological activity.2.In vitro,Wnt-S can activate the human brain microvascular endothelial cells(h CMEC/D3),human brain vascular pericytes(HBVP),human astrocytes(HA),human oligodendrocytes(HOC),mouse neural stem cells(NSC)and rat neurons(Neuron)in the Wnt/?-catenin signaling pathways,but in mice microglia(BV2)doesn't work.3.In vivo,Wnt-S can significantly promote the proliferation of neural stem cells in mouse brain.