Experimental Study On The Differentiation Of Bone Marrow Mesenchymal Stem Cells Form SAMR1 Mice Induced By Astragaloside ? Into Neurons

It has been confirmed in basic research that bone marrow mesenchymal stem cells(BMSCs)treat various neurodegenerative diseases.Astragalus membranaceus is one kind of the traditional Chinese nourishing Qi medicines.It has been proved that it can induce the differentiation of BMSCs into nerve cells.Astragaloside ? is one of the main active ingredients of Astragalus membranaceus.Whether it can promote differentiation of BMSCs into neural cells is not experimentally proven at present.The SAMR1 mice are normal senescent mice,which are control mice of the Alzheimer's disease(AD)model mice SAMP8.There are few studies on the culture and induction of differentiation of BMSCs of SAMR1 mouse.There is no report about whether they can differentiate into neurons in vitro.In this experiment,we will investigate the effect of astragaloside ? on the differentiation of bone marrow mesenchymal stem cells into neural cells in SAMR1 mice,as well as the mechanism of action.Firstly,we used whole bone marrow adherent culture method to culture BMSCs of SAMR1 mice and culture them for 3 generations.Then we used flow cytometry to detect the expression of CD29,CD34 and CD90 on the surface of cultured cells;we induced BMSCs with astragaloside ? in the concentration of 0,20,40,80,160mg/ml,and the expression of NSE and Nestin was detected by immunofluorescence at 0h,1h,6h,12 h,24h,48 h,and 72 h.We used Western blot to detect the expression of Notch-1 and ?-catenin protein during induction;Finally,we used RT-qPCR to detect the expression of Nestin,NSE,Notch-1,?-catenin mRNA after induction.The results showed that when the SAMR1 mouse BMSCs cultured to the third generation,the cells were spindle-shaped,cell size increased,basically purified.And flow cytometry detection of CD29(+),CD34(-),CD90(+),confirmed for the BMSCs.The statistical analysis of the inducing effects of different concentrations showed that the immunofluorescence of NSE and Nestin was significantly higher in the concentration of 20,40,and 80 mg/ml astragaloside ?-induced groups(T1,T2,T3)than in the control group(C)(P<0.01),and cells in the concentration of 160 mg/ml(T4)group showed death.NSE and Nestin began to express at 1h after induction.The induction effect decreased significantly after 48 h,and the induction effect was good at 6h,12 h,and 24 h,with no significant difference(P>0.05).The concentration of 40mg/ml group induced the highest expression of neurons at 12 h.In the induction after 12 h in the concentration of 40mg/ml,RT-qPCR results showed that the mRNA of Nestin and NSE was significantly higher than that of the control group(P<0.05).The expression of Notch-1 mRNA in the induction group was lower than that in the control group(P<0.05).The expression of ?-catenin m RNA in the induction group was significantly higher than that in the control group(P<0.01).In the induction after 12 h in the concentration of 40mg/ml,Western blot analysis showed that the expression of Notch-1 protein in the induced group was lower than that in the control group(P<0.01).The expression of ?-catenin protein in the induced group was significantly higher than that in the control group(P<0.01).Through experiments,we demonstrated that astragaloside ? can induce SAMR1 mouse BMSCs to differentiate into neural cells in vitro.The effect of 40 mg/ml concentration for 12 h induction is good.During the differentiation of BMSCs induced by astragaloside ?,the expression of ?-catenin protein and mRNA was increased,and the expression of Notch1 protein and mRNA was inhibited,which promoted the differentiation of BMSCs into neural cells.