Study On The Mechanism Of Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells Into Neural Cells Induced By Astragaloside ?

Bone marrow mesenchymal stem cells(BMSCs)are a kind of stem cell population concentrated in the marrow cavity,with strong regeneration activity and multidirectional differentiation ability.BMSCs transplantation has been recognized as a promising approach for the prevention and treatment of neurodegenerative diseases.Chinese traditional medicine astragalus is the traditional tonic for deficiency,which can enhance immune function,accelerate metabolism,prevent cell oxidation,remove oxygen free radicals and delay aging.It has advantages in the treatment of senile degenerative diseases.Astragaloside ? is one of the saponins of astragalus after separation and purification.It has strong biological activity and can promote the regeneration of nerve cells and protect nerve cells in vitro.?-Mercaptoethanol(?ME)is a recognized chemical method for inducing differentiation of BMSCs into neural cells.In this experiment,BMSCs of mice were extracted,cultured,purified and identified,respectively induced into nerve cells by traditional Chinese medicine and chemical inducers,and the expression of related genes and proteins were detected,providing a basis for the further application of BMSCs in the treatment of senile degenerative diseases.This study was the first to investigate the expression of related marker molecules in BMSCs induced by astragaloside ?,?ME and astragaloside ?+ ?ME,respectively.First,BMSCs were isolated aseptically in the bone marrow cavity of mice and cultured by the traditional culture method of whole bone marrow adherent.The expression of surface antigens CD29,CD34 and CD90 in the third generation cells were detected by flow cytometry to identify the purity ofBMSCs.The morphology and changes of BMSCs in each generation were observed under an inverted microscope every day,and the changes in cell activity of BMSCs were detected by MTT assay within 7 days.Then BMSCs were induced to differentiate into nerve cells by combining astragaloside ?,?ME and astragaloside ?+ ?ME,and the morphological changes of BMSCs before and after induction were observed by sem.The expression of neuron-specific enolase(NSE)and Nestin in the induced group and the control group were detected by immunofluorescence.The expressions of NSE,Nestin,Wnt3 a,Axin,?-catenin and Notch-1 genes in the induction group and the control group were detected by RT-qPCR.Western blot was used to detect the expressions of Wnt3 a,?-catenin and Notch-1 proteins before and after induction.The results showed that the newly extracted BMSCs were oval under inverted microscope,and most of the cells in the third generation were long spindle shaped after culture,passage and purification.CD29(+),CD34(-)and CD90(+)of BMSCs were determined by flow cytometry.The results of MTT assay showed that the OD value was the highest and the cell activity was the best when BMSCs were cultured to the third generation.Under scanning electron microscope,it was observed that astragaloside ?,?ME and the combined induction group of astragaloside ? could promote the differentiation of BMSCs into nerve cells,and the cell membrane of the ?ME group and the combined induction group were damaged.The results of immunofluorescence showed that the expression of NSE and Nestin after induction of astragaloside ? was higher than that of control group,?ME group and combined induction group(P<0.01).The results of RT-qPCR showed that compared with the control group,the expressions of NSE and Nestin,?-catenin,Axin and Wnt3 a genes in astragaloside ?,?ME and the combined induction group were higher(P<0.01).The expression of Notch-1 gene was lower(P<0.01).Western blot results showed that the expressions of Wnt3 a and ?-catenin in astragaloside ?,?ME and thecombined induction group were higher than those in the control group(P<0.01).The Notch-1 protein in astragaloside ?,?ME and the combined induction group was lower than that in the control group(P<0.01).In summary,we confirmed through experiments that BMSCs could be successfully differentiated into nerve cells in the astragaloside ? induction group,the ?ME induction group and the combined induction group,and the induction effect in the astragaloside ? group was better than that in the?ME induction group and the combined induction group.The mechanism of BMSCs differentiation into nerve cells induced by astragaloside ? is to activate the Wnt pathway by high expression of ?-catenin protein and gene,reduce the expression of Notch-1 gene and protein,silence the Notch pathway,and promote BMSCs differentiation into nerve cells.