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Development of analyzing softwere of neuronal death model in primary cultured cells

原代培养细胞神经元死亡模型分析软件的开发

基本信息

批准号：
07557328
负责人：
AKAIKE Akinori
金额：
$ 0.7万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

In this study, we developed the software for taking the microscopic pictures of cultured cells in a computer to automatically analyze cell viability by counting cultured cells. The automated analysis of cell viability accelerated the speed of cell-counting experiments. Using this softwere, we performed experiments to elucidate the mechanism underlying glutamate neurotoxicity in cultured rat cortical neurons and in cultured rat retinal neurons. Moreover, we searched neuroprotective factors against glutamate neurotoxicity. 1.The effect of methylcobalamin, a vitamin B^<12> analog, on glutamate neurotoxicity was examined using cortical cultures. The results suggests that methylcobalamin promotes intracellular methylation with S-adenosylmethionine, which is formed in the metabolic pathway of methylcobalamin. It is also suggested that methylcobalamin protects cortical cultures against glutamate neurotoxicity by reducing neurotoxic action of nitric oxide (NO). 2.The effect of Zn^<2+> on glutamate neurotoxicity was examined using retinal cultures. Zn^<2+> protected the cultures against glutamae neurotoxicity mediated by N-methyl-D-aspartate (NMDA) receptor. 3.The role of NO in glutamate neurotoxicity was analyzed using retinal cultures. A low concentration of NO induced a protective action against glutamate neurotoxicity by reducing the NMDA receptor-mediated currents and that elevated concentrations of NO,interacting with oxygen radicals, became toxic and enhanced glutamate neurotoxicity in the cultures. These results suggest that high concentration of glutamate released in retinal ischemia causes formation of large amount of NO,which is toxic to neurons when it is present in excess. The results in this study will offer basic reference materials to drive forward developmental research for neuroprotective drugs against many neurodegeneration diseases in central nervous system.

在本研究中，我们开发了用于在计算机中拍摄培养细胞显微照片的软件，通过对培养细胞进行计数来自动分析细胞活力。细胞活力的自动分析加快了细胞计数实验的速度。使用该软件，我们进行了实验，以阐明培养的大鼠皮质神经元和培养的大鼠视网膜神经元中谷氨酸神经毒性的机制。此外，我们还寻找了针对谷氨酸神经毒性的神经保护因子。 1.使用皮质培养物检查了甲钴胺(一种维生素B 12 类似物)对谷氨酸神经毒性的影响。结果表明，甲钴胺通过S-腺苷甲硫氨酸促进细胞内甲基化，S-腺苷甲硫氨酸是在甲钴胺的代谢途径中形成的。还表明甲钴胺通过减少一氧化氮 (NO) 的神经毒性作用来保护皮层培养物免受谷氨酸神经毒性的影响。 2.使用视网膜培养物检查Zn 2+ 对谷氨酸神经毒性的影响。 Zn 2+ 保护培养物免受N-甲基-D-天冬氨酸(NMDA)受体介导的谷氨酸神经毒性。 3.利用视网膜培养物分析NO在谷氨酸神经毒性中的作用。低浓度的 NO 通过减少 NMDA 受体介导的电流诱导针对谷氨酸神经毒性的保护作用，而高浓度的 NO 与氧自由基相互作用，变得有毒并增强培养物中的谷氨酸神经毒性。这些结果表明，视网膜缺血时释放的高浓度谷氨酸会导致大量NO的形成，NO过量时对神经元有毒。本研究结果将为推动针对多种中枢神经系统神经退行性疾病的神经保护药物的开发研究提供基础参考资料。