β-淀粉样肽诱导小胶质细胞培养上清致海马神经元损伤模型的建立

Cell Model of Amyloid β-Peptide Induced Microglia Supernatant that Damage Hippocampus Neurons

目的:建立β淀粉样肽(Aβ1-40)诱导激活小胶质细胞的上清致海马神经元损伤的细胞模型,并初步研究神经元损伤的机制。方法:用不同浓度的可溶性Aβ1-40诱导激活小胶质细胞,光镜下观察不同时间点的细胞形态,ELISA检测其分泌的肿瘤坏死因子α;用激活后的小胶质细胞条件培养基刺激海马神经元,光镜下观察细胞形态,Westernblot检测刺激后海马神经元内诱导型一氧化氮合酶(iNOS)和硝基酪氨酸(NT)的表达水平,ELISA检测海马神经元内胱冬蛋白酶-3(caspase-3)活性来评价神经元的损伤程度。结果:终浓度为10μmol/L的Aβ1-40与小胶质细胞孵育24h后,取上清液加到培养的海马神经元,孵育24～72h,海马神经元较对照组形态有明显变化;经Westernblot检测,神经元内iNOS、NT表达明显增加;ELISA检测神经元内caspase-3活性明显增高。结论:小胶质细胞被Aβ1-40激活后,其释放物有明显的致神经元损伤效应,表明建立了神经元损伤模型。

Objective： To establish the cell model of the hippocampus neurons impaired by supernatant of microglia which was induced by the amyloid β-peptide（Aβ1-40） and to study the mechanism of neurons damage. Methods： The activated microglia conditioned medium, supernatant of activated microglia stimulated by Aβ1-40 was used to stimulate the hippocampus neurons. The morphology of cells was observed at different time points by light microscopy and the expression of TNFα was detected by ELISA. To evaluate the neurons injuried by Aβ1-40 the morphology of cells was observed by light microscopy, the expression of inducible nitric oxide synthase（iNOS） and nitrotyrosine（NT） in stimulated hippocampus neurons was detected by Western blot, the activity of caspase-3 of the hippocampus neurons was detected by ELISA. Results： The microglia were incubated with Aβ1-40（final concentration of 10μmol/L） for 24h, then the supernatant was added to cultured hippocampus neurons and incubated for another 24-72 h, compared with the control group, the hippocampus neurons were damaged severely by the supernatant of activated mieroglia, and their morphology had a significant change. The expression of iNOS and NT and the activity of caspase-3 in hippocampus neurons were significantly increased. Conclusion： We established a model of neuron damage successfully. After activated by Aβ1-40, microglia can release factors which have significant damag-effect on neurons.