| Background:Alzheimer's disease,the commonest cause of dementia,is a growing global health concern with huge implications for individuals and society.The exact cause of Alzheimer's disease is unknown and is usually caused by a combination of multiple genetic,familial and environmental factors,with age,family history and genes being major risk factors.Alzheimer's disease is a progressive disease with no known cure.In recent years,stem cell transplantation has attracted more and more attention as a new method for the treatment of neurodegenerative diseases.Bone Marrow mesenchymal stem cells(BMSCs)are a good type of transplanted stem cells,which are available in large amounts,harvested directly through simple procedures,immune-tolerant and unrestricted in moral and ethical aspects.Current studies have found that bone marrow mesenchymal stem cells have the function of neural differentiation,but they are more differentiated into glia and less differentiated into neurons.Neurotrophic factors play an important role in the normal development and function of the nervous system.One of them,neurotrophin-3(NT-3),has been shown by increasing evidence to promote neuronal survival and nerve repair.It has been found that NT-3 can induce BMSCs to differentiate into neurons,and transplantation of overexpressed NT-3 BMSCs can promote the recovery of motor function and nerve regeneration in the rat model of spinal cord injury.Therefore,we speculate that NT-3 gene can more effectively promote neuron-like differentiation of BMSCs,and transplantation of NT-3 gene modified BMSCs may play a certain role in neural regeneration and improvement of cognitive function.Objectives:This study aims to explore the role of NT-3 in promoting neuron-like differentiation of BMSCs in vivo and in vitro and its possible mechanism,and to explore the improvement of cognitive function of NT-3-modified BMSCs transplantation in Alzheimer's disease rats,so as to provide certain experimental and theoretical basis for BMSCs transplantation in the treatment of Alzheimer's disease.Methods:This study consists of in vitro experiment and in vivo experiment:Part ?:In vitro experiment1.After Sprague-Dawley(SD)rats were killed,femoral and tibial bone marrow were separated under aseptic conditions,bone marrow cells were collected,subcultured to the third generation of BMSCs,and BMSCs were identified by flow cytometry with CD90,CD44,CD45,and CD34 antibodies.BMSCs were induced osteogenic differentiation and adipogenic differentiation,respectively,and identified by staining.2.The synthetic rat NT-3 full-length gene was cloned into a lentiviral expression vector,and the expression of NT-3 was silenced with short hair pin RNA(sh-RNA).NT-3 overexpression lentivirus and sh-NT-3 lentivirus were constructed,and the expression level of NT-3 was detected by Western Blot to determine the lentivirus with the best interference efficiency.3.BMSCs were transfected with lentivirus.BMSCs were infected with concentrated virus and 6?g/mL polyene in serum-free medium for lentivirus-mediated transduction.After 24h,complete culture medium was used to replace the medium,and the expression level of NT-3 was detected by Western blot.4.Newborn SD rats were taken for the isolation,culture,purification and purity identification of primary neurons.Original generation of neurons and the third generation of BMSCs trained 8 days,the immune cells in chemical experiments to detect neuronal markers beta ?-tubulin/beta-catenin,the expression of NSE and NF-200,determined by MTT 24 h,48 h and 72 h was detected in the cell vitality,flow cytometry to detect each cell cycle and cell apoptosis.5.The expression of ?-catenin in the whole cell protein and in the nucleus were detected by Western Blot.The expressions of NT-3 and ?-catenin in each group were detected by fluorescence quantitative PCR and immunofluorescence.The second part is in vivo experiments1.The AD rat model was established by injecting oligomer A?1-42 into the cerebral ventricle of rats.SD rats were randomly divided into four groups,including PBS group,BMSCs group,BMSCs group with overexpression of NT-3,and BMSCs group with silenced NT-3.Seven days after the establishment of AD model,PBS,BMSCs,overexpressed NT-3 BMSCs and silenced NT-3 BMSCs were injected into the bilateral hippocampus of AD rat model by stereotactic method.One month later,Morris water maze method was used to observe and evaluate the behavioral changes during space exploration test and positioning sea trial.The cognitive function of rats was measured by new object recognition experiment.2.One month after BMSCs transplantation,after the behavior test,the hippocampus of rats was collected and Western Blot was used to detect NT-3,Synapsin-1 and Synaptophysin.SYP),postsynaptic densitin-95(PSD95),whole protein ?-catenin,and nuclear ?-catenin expression.3.One month after BMSCs transplantation,the expression of A?1-42 was detected by immunohistochemistry and the expression of NSE and NF-200 was detected by immunofluorescence.Results:Part ?:In vitro experiment1.The surface markers of the third-generation BMSCs were detected by flow cytometry,and the results showed that CD90 and CD44 were strongly positive on the cell surface,while CD45 and CD34 were negative on the hematopoietic stem cell markers.2.BMSCs were successfully transfected with lentivirus,and NSE,??-tubulin and NF-200 were positive by immunofluorescence staining.After transfection,cell viability was high,proliferation was strong and apoptosis rate was low.3.Real-time PCR showed that overexpression of NT-3 significantly increased the mRNA expression of ?-catenin in BMSCs.Western Blot results showed that the expression levels of total protein and ?-catenin protein in the nucleus of co-cultured BMSCs were significantly increased,and the expression levels of NT-3 gene were significantly down-regulated by silencing.The expression of ?-catenin was detected by immunofluorescence staining,which further confirmed that the overexpression of NT-3 could promote the expression of ?-catenin in BMSCs,and the silencing of NT-3 could reduce the expression.Part ?:In vivo experiments1.After transplantation of BMSCs in AD rats,the cognitive function and memory ability of NT-3-BMSCs group were significantly improved compared with PBS group;The cognitive function and memory ability of rats in sh-NT-3-BMSCs group were significantly worse than those in NT-3-BMSCs group.2.One month after BMSCs transplantation,the expression of NT-3 protein in hippocampus was detected by Western Blot.The expression of NT-3 protein in hippocampus of rats in NT-3-BMSCs group was significantly increased compared with that in PBS group.The expression of NT-3 in sh-NT-3-BMSCs group was significantly lower than that in NT-3-BMSCs group.3.Immunohistochemical results showed that the level of A?1-42 in NT-3-BMSCs group was significantly lower than that in PBS group;the level of A?1-42 in sh-NT-3-BMSCs group was significantly higher than that in NT-3-BMSCs group.4.Western Blot results showed that the level of NT-3 in the hippocampus of NT-3-BMSCs group was significantly higher than that of PBS group,and the level of NT-3 in sh-NT-3-BMSCs group was significantly lower than that of NT-3-BMSCs group.The levels of Synapsin-1,SYP and PSD95 in NT-3-BMSCs group were significantly higher than those in PBS group.The synapsin-1,SYP and PSD95 levels of SH-NT-3-BMSCs were significantly lower than those of NT-3-BMSCs.The levels of total protein and intracellular protein ?-catenin in NT-3-BMSCs group were significantly increased compared with that in PBS group,while the levels of total protein and intracellular protein ?-catenin in sh-NT-3-BMSCs group were significantly decreased compared with that in NT-3-BMSCs group.5.The expression of NSE and NF-200 was detected by immunofluorescence staining.Compared with the PBS group,the expression of NSE and NF-200 was significantly increased in the NT-3-BMSCs group.Compared with NT-3-BMSCs group,the expression of NSE and NF-200 in sh-NT-3-BMSCs group was significantly decreased.Conclusion:1.Lentivirus mediated NT-3 gene can successfully transfect BMSCs,and BMSCs can stably express NT-3 gene.2.NT-3 can successfully and stably promote neuron-like differentiation of BMSCs.3.NT-3 promotes neuron-like differentiation of BMSCs through activation of the Wnt/?-catenin signaling pathway.4.NT-3-modified BMSCs can improve the cognitive function of dementia rats.5.NT-3 promotes neuron-like differentiation and nerve regeneration of BMSCs through activation of Wnt/?-catenin signaling pathway,and improves cognitive function of dementia rats. |
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