| Alzheimer’s disease (AD) is a degenerative disease of nervous system, it severely damages the life quality of the middle-aged and elderly people. As the advent of global population ageing, the number of the AD patients increases year by year, and the harm to society increases still. AD leads to the progressive and irreversible decline in memory, cognition and ability to learn, the patients gradually lose self-care ability. Most of the patients eventually die of infection. The pathogenesis of AD is still unclear, but the importance of amyloid β in AD is accepted by most researchers. Aβ is produced excessively during AD, and become Aβ oligomers and Aβ amyloid fibrils. They gradually deposit as senile plaques. The toxic senile plaques is the most important pathological characteristics of AD. As the complexity, the researchers still couldn’t explain the mechanism of Aβ neurotoxicity. Therefore, the study of Aβ neurotoxicity is a hot spot of research, and it’s of great significance for the diagnosis and treatment of AD.To study the molecular mechanism of Aβ induced neuron injury, Neuro-2a and cerebral cortex neurons of embryonic mice are used to establish the cell model. Diffirent methods are used, including MTT technics, observing in microscope, Hoechst fluorescent staining and TUNEL staining, to study the influence of Aβ25-35 on the neuron cells. The results shows that there’s dose-dependent toxic effect of Aβ25-35 on the cells. Aβ25-35 can cause morphology changes in N2a cells, including cell differentiation, cell shrinking and cell debris. And changes in cerebral cortex neurons, such as synapses broken, cell becoming smaller and shrank. The experiment also shows apoptosis cells increasing by Aβ25-35. These results show that Aβ25-35 can cause damage to neuron cells.According to many studies, the released nucleotides in the brain injury could be involved in mediating cell injury, and extracellular nucleotides include ATP, ADP, UTP, UDP and their derivative. To clear the role of extracellular nucleotides in Aβ induced cell injury, we add nuclease Apyrase into the culture medium before the Aβ25-35 stimulation. It shows that the addition of Apyrase recover the N2a cell viability. So the extracellular nucleotides may be involved in the neuron cell injury. Further more, we add different concentrations of ATP, ADP, UTP, UDP into N2a cells. The MTT assay shows that only 3,5,10 mmol/L ATP could cause a significant concentration-dependent cell viability decline in N2a cells. Observing in microscope, Hoechst fluorescent staining and TUNEL staining results also confirmed this phenomenon. Then we detect the extracellular ATP in the supernatant using ATP bioluminescent assay. The results show that Aβ25-35 could cause time-dependent release of ATP from N2a and N9 cells.We found that the expression of P2X7 which is regarded as the receptor of ATP elevated after treating N2a cells with Aβ25-35 by quantitative real time PCR. Then, we used P2X7 receptor inhibitor BBG to pretreat neuron cells, the results show that the cell injury caused by Aβ25-35 and ATP was significantly relieved. Extracellular ATP may play an important role in regulating neuron cell injury by binding to its receptor P2X7. Finally, evidences which is obtained by quantitative real time PCR and Western blot indicated that released ATP induced by Aβ could impair neuron cells viability through increasing BAX expression and activating Caspase-3 apoptotic signaling pathway.Above all, Aβ25-35 can cause ATP release, and then extracellular ATP bind to neural cytoplasm membrane P2X7 receptor, inducing the activation of apoptosis related proteins, such as BAX, Caspase-3, which finally induce neuron cells apoptosis. The inhibitor of P2X7 receptor BBG could partially block Aβ25-35 induced neuron cell injury. This thesis shows a new strategy for studying AP’s cytotoxicity, and it may provide a theoretical basis for P2X7 receptor as the target for treatment of AD. |
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