梓醇保护L谷氨酸与Aβ2535损伤PC12细胞作用比较.docVIP

梓醇保护L谷氨酸与Aβ2535损伤PC12细胞作用比较 　　【摘要】 目的 观察梓醇对L-Glu和Aβ25-35损伤PC12细胞的保护作用的异同。方法 常规培养神经元样PC12细胞，预先加入梓醇及对照生理盐水24 h后分别加L-Glu 5 mmol/L和Aβ25-35 20μmol/L损伤24 h，K252a 干预组细胞在梓醇前1 h加入200 nmol/L K252a。MTT法测定细胞存活率，放射配基结合分析测定M2受体密度。结果 梓醇100 μmol/L明显提高细胞存活率(P25-35 损伤细胞的保护作用，对梓醇保护L-Glu损伤的阻断作用更强。结论 梓醇对L-Glu和Aβ25-35损伤PC12细胞保护作用有差异。 　　【关键词】 梓醇；PC12细胞；L-Glu；Aβ25-35；K252a 　　Compare of the protective effect of catalpol on PC12 cells injury induced by L-glutamate and Aβ25-35 WANG Jin-hong,ZHU Su-hua,SUN Shan-ming,et al.Department of Pharmacology, Weifang Medical College, Weifang 261042, China 　　【Abstract】 Objective AimTo observe the difference between effect of catalpol on PC12 cells injury induced by L-glutamate and Aβ25-35. Methods Neuron-like PC12 cells were cultured, 24 h after cells were exposed to catalpol 10 μmol/L,100 μmol/L and saline control respectively. L-Glutamate 5 mmol/L and Aβ25-35 20 μmol/L was added another 24 h later and stayed in DMEM for 24 h. When necessary, 200 nmol/L K252a was added 1 h before catalpol addition. Cell viability was measured by MTT assay, M2 receptor density was measured by single point 3 h-QNB binding assay. Results Catalpol elevated cell viability at the final dose of 100 μmol/L(P25-35 but blocked the effect on cells injured by L-glutamate more significant. Conclusion There were some difference between the protective effect of catalpol on PC12 cells injured by L-glutamate and Aβ25-35 respectively. 　　【Key words】 Catalpol; PC12 cell; L-glutamate; Aβ25-35；K252a 　　 　　梓醇是滋阴药地黄的主要活性成分，近年研究表明其有显著神经保护作用[1-3]。我们前期研究发现梓醇保护Aβ25-35 损伤PC12 细胞及脑内注射Aβ25-35 拟痴呆小鼠的胆碱能神经系统功能，保护兴奋性神经递质谷氨酸(glutamate, Glu)对细胞的神经毒性，显著改善老年痴呆的病理变化。研究中发现梓醇对Aβ25-35和L-Glu损伤PC12细胞的保护作用不同，现将结果报告如下。 　　 　　1 材料与方法 　　 　　1．1 主要试剂 鲜地黄采于河南省温县。梓醇从鲜地黄经乙醇和正丁醇提取，硅胶柱分离，重结晶纯化制得，经高效液相色谱和质谱鉴定，纯度95%。DMEM (Gibco ,USA)，Aβ25-35、L-Glu、K252a、四甲基偶氮唑蓝(methyl thiazolyl tetrazolium，MTT)(sigma ,USA)，3 h-QNB(Amersham 公司，比活度1．54 TBq/ mmol)。Aβ25-35 用灭菌双蒸水溶解后配成100mol/L母液无菌分装，-20℃储存，临用前37℃孵育3~5 d使成为聚集状态[4]，加入DMEM稀