Neurotrophin-3 Contribute To Neuronal Differentiation Of Bone Marrow Mesenchymal Stem Cells Through The Activation Of The C/EBP ? Pathway

Background:Alzheimer disease(AD),the neurodegenerative diseases of the central nervous system is one of the urgent problems that has troubled the medical profession so long time.Cognitive impairment,the dominant clinical manifestation of Alzheimer disease(AD),seriously affects the health and the quality of life of middle-age and aged people.Bone marrow mesenchymal stem cells(BMSCs)are multipotent stem cells derived from bone marrow and have become a good source of stem cells transplantation,characterized by abundant source,slighter wound,immune tolerance,and no restrictions of morality and ethics.In recent years,Treatment of AD with BMSCs has dawn more attention.Purpose: In this study,we established transgenic BMSCs overexpressing NT-3 by lentivirus(plenti-NT-3)and transplanted these cells into the hippocampus of SD rats model of Alzheimer ' s disease(AD).We investigats the effects and mechanisms of NT-3-BMSCs Transplantation in hippocampus of AD rats model.Method:1.The bone marrow mesenchymal stem cells(BMSCs)of SD rats was isolated and cultured in vitro by density gradient centrifugation.The morphology was observed under inverted microscope.The BMSCs surface markers CD44,CD90,hematopoietic cell markers CD45,CD34 of BMSCs were detected by flow cytometry.2.We transfected BMSCs with Lentivirus-NT-3 for 48 hours,and the expression of NT-3protein were detected by western blot(WB)method.NT-3 overexpression of lentivirus and inhibition of lentivirus transfected BMSCs cells,followed by cultured with neurons.MTT was used to detect the proliferation of BMSCs after co cultured with neurons cells at 24 h,48 h and 72 h.The C/EBP beta mRNA expression was detected by real-time fluorescent quantitative PCR.The C/EBP beta protein expression was detected by Western blot.3.The AD model was set up by injection with beta-amyloid peptide 1-42(A?1-42)into the hippocampus of both sides bilatelly.The AD rats were selected and then divided into three groups randomly,and those were PBS group,BMSCs group,NT-3-MSCs group.The AD models were treated by PBS,BMSCs,NT-3 gene modified BMSCs respectively,those ADmodels were transplanted through hippocampus of both sides after 7 days of model establishment.The ethological changes of the AD rat model were observed by Morris water maze experiment after 1 month of transplantation,4.The expression of NT-3 protein and C / EBP? in both hippocampus of rat were detected by Western Blot after 1 month of transplantation.The expression of ??-tubulin in hippocampus were detected by immunofluorescence.Result:1.The expression of BMSCs markers was highly positive for CD44(91.2%)and CD90(90.8%),but negative for CD34(2.3%)and CD45(2.9%).2.Western Blot shows the NT-3 expression in BMSCs was observed after the constructed lentivims infected BMSCs.The results of MTT showed that compared with the control group,the proliferation of BMSCs cells was significantly enhanced in the NT-3overexpressing group at 48 h and 72 h(P < 0.05).Real-time PCR and Western Blot results showed that C/EBP beta mRNA and protein levels were significantly up-regulated in NT-3overexpressing BMSCs cells,but significantly decreased in NT-3 silencing group BMSCs(P< 0.05).3.The learning memory score of the rats in BMSCs group and NT-3-BMSCs group increased comparing with PBS,The NT-3-BMSCs group has a significant increase comparing with with PBS(P < 0.05).4.Compared with PBS group,the expression level of NT-3 and C / EBP ? of BMSCs group and NT-3-BMSCs group in the hippocampus were increased.NT-3-BMSCs group has a significant increase comparing with with PBS(P < 0.05).5.Immunofluorescence staining showed that compared with PBS group,the expression of ??-tubulin increased significantly in hippocampus of BMSCs group and NT-3-BMSCs group,especially the NT-3-BMSCs group.Inconclusion:1.The cells selected by density gradient centrifugation ability which had basic phenotypical properties of MSCs,they have good activity and high purity by subculturing.NT-3 gene-modified BMSCs which could express NT-3 abundantly.2.The transplanted NT-3 modified BMSCs or BMSCs could improve the learning andmemory ability of AD rats.The overexpression of NT-3 could promote neuronal differentiation and nerve regeneration in hippocampus of AD rats.It may be mediated by activating transcription factor C / EBP ? Subtype LAP to achieve.