The Effects Of BDNF Gene-modified Neμral Stem Cells On The Expression Of ERK1/2 And PSD-95 In Aβ_25-35-injured PC12 Neurons

Objective: In this thesis, the Alzheimer disease mode was constructed with PC12 nerve cell which is vulnerated by Aβ_25-35.Nerve cellswas differentiated from BDNF neural stem cells, which was then cultivated with PC12 nerve cell to discover whether nerve synapse-like body cultivated with brain-derived neurotrophic factor BDNF could block neuron damage induced by Aβ_25-35.Methods: PC12 nerve cell was differentiated to neuron-like cell induced by NGF, which was then vulnerated for different minutes by Aβ_25-35 in different concentrations, and the survival rate of nerve cell was tested through CCK8 assay to screen out early stage Alzheimer disease mode; Morphological change of PC12 nerve cells was observed through immunofluorescence technique; Expression of erk1/2 and PSD-95 of PC12 was tested through Western blot assay. Neural Stem Cells was infected in vitro by Ad-BDNF-EGFP to screen out cells which can express BDNF and EGFP gene stably, which was then cultivated with PC12 nerve cell vulnerated by Aβ_25-35, and the expression of m RNA and protein of erk1/2 and PSD-95 in control, mode, BDNF-NSCs 72 h group, EGFP-NSCs72 h group, BDNF-NSCs96 h group and EGFPNSCs96 h group was tested through Quantitative Real-time PCR and Western bolt assay. The cultivated synaptic connectivity was observed through immunol staining assay.Results:1. Oligomer Aβ_25-35 can reduce the survival of PC12 nerve cell dose dependently. Among them, The difference of 5μmol/L 、 10μmol/L 、 20μmol/L Aβ_25-35 group was significant compared with control group. The apoptosis rate is remarkable when PC12 nerve cell was treated with10μmol/L Aβ_25-35 for 48h. It is even worse when the concentration is 20μmol/L.2. The result of immunofluorescence from nerve granule and neuregulin suggested that Neurotoxicity characteristics, such as shorter Synaptic length,shrinking of Pericaryon and loosenedneural network,of PC12 nerve cellin morphologywas observed significantly after treated for 48 h with 10μmol/L Aβ_25-35.3. The western blot assay further suggested that the expression of erk1/2 and PSD-95 decreased after treated 48 h with 10μmol/L Aβ_25-35, and the difference is significant when compared with control group. Therefore, the PC12 nerve cell treated with 10μmol/L Aβ_25-35 can be considered as a good AD cell mode.4. Both Quantitative Real-time PCR and Western blot assay suggested that nerve cell derived from Neural Stem Cells can construct synaptic connectivity with PC12 cell, and protect PC12 from neuronal damage derived from Oligomer Aβ_25-35. The expression of erk1/2 m RNA, PSD-95 m RNA and erk1/2 protein PSD-95 protein was increased.5.Immunol staining assay further proved that, when cultivated for 96 h, neural stem cells infected by BDNF connected much closer than that of 72 h, and the brunches are much more and longer.Conclusion: 1. The most effective concentration to ensure early stage AD mode is 10μmol/L and time 48h. 2. The damage of Oligomer Aβ_25-35 to nerve cell can lead to the shrinking of neuronal cell body,loosenedneural network and decrease of erk1/2 protein and PSD-95 protein. 3. BDNF can repair synapsis and protect nerve cell by inhibiting the decrease of erk1/2 protein and PSD-95 protein derived from Oligomer Aβ_25-35.