Effects And Mechanisms Of Thrombospondin-4(THBS-4) On NG2~+ Cell Differentiation In Vitro

BackgroundNG2 cells belong to glial precursor cells and are widely distributed in the central nervous system.They proliferate extensively after central nervous injury and participate in the formation of glial scars.With a variety of differentiation potential,recent studies have shown that NG2 cells have the potential to differentiate into neurons.This provides potential cellular resources for the treatment of neurological diseases.Thrombospondin-4(THBS4)is a member of the thrombospondin family.THBS4 plays an important role in regulating nervous system development and signal transduction,but the role of THBS4 in NG2 cell differentiation and the mechanism is still unclear.This study used the gene silencing and gene overexpression technology to study the effect of THBS4 gene on the differentiation of NG2 cells in vitro,and to reveal the role of THBS4 in the reprogramming of NG2 cell fate,and provide a new cell therapy for neurodegenerative diseases.Objective1.To eludicate of the role of THBS4 in NG2 cell reprogramming;2.To determing which transcription factors are involved in the process of NG2 cell reprogramming.Methods1.The effect of THBS4 on the differentiation of NG2 cells into neurons.NG2 cells were infected with overexpressing viral plasmid of THBS4,and the expression levels of neuronal markers were detected by Western Blotting and Real-time PCR;siRNA was used to interfere with the expression of TNBS4 in NG2 cells,and the expression levels of neuronal markers were detected by Western Blotting and Real-time PCR.2.The effect of THBS4 on the differentiation of NG2 cells into glial cellsNG2 cells were infected with overexpressing viral plasmid of THBS4,and the expression levels of oligodendrocytes and astrocyte markers were detected by Western Blotting and Real-time PCR.siRNA was used to interfere with the expression of THBS4 in NG2 cells,and the expression levels of oligodendrocytes and astrocyte markers were detected by Western Blotting and Real-time PCR.3.Which transcription factor is regulated by THBS4NG2 cells were infected with overexpressing virus plasmid of THBS4,and the xpression levels of transcription factors Ngn1,ATF6 and Olig2 were detected by eal-time PCR;siRNA was used to interfere with the expression of THBS4 in NG2 cells,and he expression levels of transcription factors Ngn1,ATF6 and Olig2 were detected y Real-time PCR;Results1.To investigate the effect of THBS4 on neuronal reprogramming of NG2 cells,we overexpressed THBS4 in NG2 cells and found that the expression levels of neuronal markers Tuj1 and MAP2 increased with the increase of THBS4 expression level.The expression of THBS4 in NG2 cells was interfered by siRNA,and the results were reversed;This suggests that THBS4 can promote reprogramming of NG2 cells to neurons.;2.To investigate the effect of THBS4 on the reprogramming of NG2 cells to glial cells,we overexpressed THBS4 in NG2 cells and found that the expression levels of oligodendrocytes and astrocytes markers increased with the increase of THBS4 expression level.The expression of THBS4 in NG2 cells was interfered by siRNA,and the results were reversed;This indicates that THBS4 can inhibit the reprogramming of NG2 cells intoglial cells.3.In order to verify which transcription factor plays a role in NG2 cell reprogramming,we overexpressed THBS4 in NG2 cells and found that the expression level of transcription factor Ngn1 increased.Interfering with the expression of THBS4 in NG2 cells,the results were reversed.This indicates that THBS4 plays a role in the reprogramming of NG2 cells to neurons by activating the transcription factor Ngn1.Conclusion1.THBS4 promotes the differentiation of NG2 cells into neurons.2.THBS4 inhibits the differentiation of NG2 cells into oligodendrocytes and astrocytes.3.THBS4 regulates this process by activating the transcription factor Ngn1...