山西医科大学 硕士学位论文 代谢型谷氨酸受体调控-APP表达在铝神经毒性中的作用 姓名：杨焱 申请学位级别：硕士 专业：劳动卫生和环境卫生学 指导教师：牛侨 2010-05-25 山西医科大学硕士学位论文 I 代谢型谷氨酸受体调控代谢型谷氨酸受体调控代谢型谷氨酸受体调控代谢型谷氨酸受体调控-APP-APP-APP-APP 表达在铝神经毒性中的作用表达在铝神经毒性中的作用表达在铝神经毒性中的作用表达在铝神经毒性中的作用 中中中中 文文文文 摘摘摘摘 要要要要 目的目的 通过染铝体外神经细胞培养，研究-APP在铝致神经细胞丢失与铝致神经毒性 的关系出发，探讨代谢型谷氨酸受体与铝致神经毒性的关系，以及-APP和代谢型谷氨酸 受体的关系。方法方法 神经细胞原代培养并用AlCl36H2O染毒，使铝的终浓度分别为 0mmol/L、0.5mmol/L、1mmol/L 、2mmol/L。作用48h后相差显微镜观察细胞的生长状态， 应用Cell Counting Kit-8检测各组神经细胞的细胞活力，AO-EB和Hoechst染色法观察铝 染毒细胞的存活和凋亡；流式细胞仪AnnexinV-PI双染法定量测定细胞的凋亡率；用荧光 定量PCR和免疫组化检测神经细胞-APP、mGluR1、mGluR3、 mGluR7的表达。对培养的 原代神经细胞用2mmol/L铝染毒，加入不同浓度的代谢型谷氨酸受体激动剂或拮抗剂，检 测细胞的活力、流式细胞仪Annexin V-PI双染法定量测定细胞的凋亡率,用荧光定量PCR和 免疫组化检测神经细胞-APP、mGluR1、mGluR3、mGluR7的表达。结果结果 染毒可使细胞 突起萎缩,细胞胞体增大变圆、细胞数量减少,并且细胞界限不清;同时病理改变随着Al3 + 浓度增加和染毒时间延长而加重；AO-EB和Hoechst染色法显示了铝染毒细胞的凋亡和坏死 的形态学变化；神经细胞活力试验结果：各组神经细胞活力差异有统计学意义（P0.01）， 0.5mmol/L、1mmol/L 、2mmol/L Al3+剂量组神经细胞的活力低于0mmol/L Al3+组；流式细 胞仪定量检测结果：各组神经细胞凋亡率差异有统计学意义（P0.01），1mmol/L Al3+ 和2mmol/L Al3+剂量组神经细胞的凋亡率显著高于0mmol/L Al3+组；荧光定量PCR和免疫组 化检测神经细胞结果显示：与对照组相比，低、中剂量组mGluR1、mGluR3 、mGluR7的基 因和蛋白表达尚未达到统计学意义（P0.05），高剂量组mGluR1的基因和蛋白表达升高， 差异有统计学意义（P0.01）；与对照组相比，高剂量组mGluR3、 mGluR7基因和蛋白表 达下降，差异有统计学意义（P0.05），中、高剂量组-APP的基因和蛋白表达均升高，差异 有统计学意义（P0.05）；加入、组代谢型谷氨酸受体激动剂可以显著改善染铝后 的神经细胞坏死的形态学改变，加入、组代谢型谷氨酸受体拮抗剂可以增加染铝后的 细胞坏死的形态学改变；不同浓度代谢型谷氨酸受体激动剂可以显著提高神经细胞活力 (P0.05, P0.01)，不同浓度代谢型谷氨酸受体拮抗剂可以显著降低细胞活力(P0.05, P0.01)；流式细胞仪测定结果显示：、组代谢型谷氨酸受体激动剂可以显著降低神 经细胞的凋亡率 (P0.01)，、组代谢型谷氨酸受体拮抗剂可以显著提高神经细胞的 凋亡率 (P0.01)；荧光定量PCR和免疫组化结果显示：与染铝对照组相比，、组代谢 型谷氨酸受体激动剂可以显著降低染铝神经细胞的mGluR1、mGluR3 、mGluR7和-APP的 基因和蛋白表达。结论结论 1.铝能够诱导神经细胞的凋亡，其凋亡程度与铝的作用剂量有密 切关系。2.随着染铝的浓度的增高-APP表达增高，加入、组代谢型谷氨酸受体激动 剂-APP表达下降，提示：-APP在铝致神经细胞丢失与铝致神经毒性有关；-APP与代 山西医科大学硕士学位论文 II 谢型谷氨酸受体有关。3.铝诱导mGluRs表达异常：mGluR1表达升高，mGluR3 mGluR7表达 下降。提示代谢型谷氨酸受体与铝致神经毒性有关。4.、组代谢型谷氨酸受体激动剂 具有神经保护作用。 【关键词】铝神经细胞-APP神经毒性mGluRs 山西医科大学硕士学位论文 III RegulationRegulationRegulationRegulation ofofofof metabotropicmetabotropicmetabotropicmetabotropic glutamateglutamateglutamateglutamate receptorreceptorreceptorreceptor -APP-APP-APP-APP expressionexpressionexpressionexpression inininin thethethethe roleroleroleroleofofofof aluminumaluminumaluminumaluminum neurotoxicityneurotoxicityneurotoxicityneurotoxicity A A A Abstractbstractbstractbstract ObjectiveObjectiveObjectiveObjective Aluminum exposure in vitro by cultured neurocytes to study the -APP induced neurocytes loss in aluminum and aluminum neurotoxicity induced by the relation, of metabotropic glutamate receptors and the relationship between the neurotoxicity induced by aluminum, and -APP and metabotropic glutamate receptors.MethodsMethodsMethodsMethods: primary cultured neurocytes with AlCl3 6H2O exposed to final concentrations of aluminum were 0mmol / L, 0.5mmol / L, 1mmol / L, 2mmol / L. 48h post-contrast microscope observation of the role of cell growth state, application Cell Counting Kit-8 group of neurocytes was detected by cell viability, AO-EB and Hoechst staining of aluminum exposure cell survival and apoptosis; flow cytometry AnnexinV-PIdouble staining quantitative determination of apoptosis rate; using fluorescence quantitative PCR and immunohistochemical detection of neurocytes -APP,mGluR1, mGluR3, mGluR7 expression. The cultured primary neurocytes with 2mmol/L of aluminum were exposed to different concentrations of metabotropic glutamate receptor agonist or antagonist, to detect the cell viability, flow cytometry, AnnexinV-PI double staining quantification apoptosis rate determined by fluorescence quantitative PCR and immunohistochemical detection of neurocytes -APP,mGluR1, mGluR3, mGluR7 expression.Results:Results:Results:Results: exposed cell processes could shrink, increasing the cell body rounded, cells decreased, and the cell boundaries unclear; the same time as the pathological changes of Al3 +concentration and exposure time of prolonged; AO-EB and Hoechst staining method shows the aluminum exposed cells morphological changes of apoptosis and necrosis; neurocytes viability test result: The neurocytes activity was significantly (P 0.01), 0.5mmol / L, 1 mmol / L, 2mmol /LAl3+ activity of neurocytes dose group than 0mmol /LAl3 +group; flow cytometry test results: The group of neurocytes apoptosis rate was significantly (P 0.05), high dose group mGluR1 gene and protein expression increased significantly (P 0.01); compared with the control group, high dose group mGluR3, mGluR7 gene and protein decreased significantly (P 0.01); compared with the control group, middle and 山西医科大学硕士学位论文 IV high dose group -APP gene and protein expression increased significantly (P 0.05); joined Group , metabotropic glutamate receptor agonist, significantly improved after the neurons of aluminum-like morphological changes of necrosis, joined the Group , metabotropic glutamate receptor antagonist, aluminum can be increased after transfection The morphological changes of cell necrosis; different concentrations of metabotropic glutamate receptor agonists can significantly im