Neuroprotection for Cell Death of Retinal Ganglion Cells and Molecular Biological Study for Nervi-Regeneration

视网膜神经节细胞细胞死亡的神经保护和神经再生的分子生物学研究

基本信息

批准号：
11470364
负责人：
MISHIMA Hiromu
金额：
$ 8.38万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470364/
关键词：
retinal ganglion glutamate neurotoxity intracellular Ca^<2+> concentration glutamate receptor cDNA microarray novel gene nitric oxide nitric oxide synthase 網膜 differential display

项目摘要

NMDA, AMPA and kainate receptors were confirmed to exist on MACS-separated cultured RGC. Moreover, 20-HE inhibited NMDA receptor-mediated currents most prominently and AMPA- and kainate-mediated currents moderately.cDNA arrays were used to detect highly expressed mRNA in the mouse eye. We focused among them on a novel gene, ODAG (GenBank ; Accession No.AB047921), which was downregulated at P10. Mouse ODAG cDNA encodes a protein of 266 amino acids. Human ODAG cDNA and genomic structure were identified by BLAST analysis of the GenBank database with mouse ODAG. Mouse ODAG-specific mRNA expression was detected in various mouse tissues within the eye at P2 and P7, whereas it was not detected anywhere at P14.Expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) induced by glutamate stimulation were investigated. Immunohistochemical analysis revealed nNOS and eNOS expressed in r … More etinal ganglion cells. Intracellular NO levels in cultured RGCs showed fluctuation during a 20-minute observation. The presence of a specific nNOS inhibitor significantly inhibited the increase of intracellular NO after the introduction of glutamate to fee medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs, and demonstrated that NO is produced by nNOS mainly that is stimulated by glutamate in cultured RGCs.Glutamate caused cell death to retinal ganglion cell for 24 hrs in a concentration-dependent manner. Glutamate increased in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in a concentration-dependent manner. NMDA and KCl caused the same results. Betaxolol inhibited the KCl-induced increase in [Ca^<2+>]_i to 25 %. On the other hand, betaxolol significantly inhibited glutamate-induced RGCs death. An increase in [Ca^<2+>]_i is considered the first signal of glutamate neurotoxicity. Betaxolol produced the neuroprotective effects by inhibiting voltage-dependent calcium channels. This method is probably useful for searching neuroprotective drugs and new glaucoma therapy. Less

NMDA，AMPA和海藻酸盐受体已证实存在于MAC分离的RGC上。此外，最突出的20-he抑制NMDA受体介导的电流和AMPA-和Kainate介导的电流中等。CDNA阵列用于检测小鼠眼中高度表达的mRNA。我们在其中专注于一个新的基因ODAG（GenBank；登录号AB047921），该基因在P10下被下调。小鼠ODAg cDNA编码266个氨基酸的蛋白质。通过用小鼠ODAG对GenBank数据库的爆炸分析来鉴定人ODAg cDNA和基因组结构。在P2和P7的眼睛内的各种小鼠组织中检测到小鼠ODAG特异性mRNA表达，而在p14中未检测到它的任何地方。一氧化氮合酶的同工型（NOS）的表达，无需生产的酶，由大鼠氧化物（NO）在大鼠网状细胞中的合成（no）刺激（rgc）rgcs（rgcs）rgcs的含量（no）。免疫组织化学分析揭示了在r…更神经节细胞中表达的NNO和eNOS。在20分钟观察过程中，培养的RGC的细胞内无水平显示出波动。特定的NNO抑制剂的存在显着抑制了将谷氨酸引入费用培养基后细胞内NO的增加。这项研究表明，所有组成型NOS同工型均在RGC中表达，并证明NNOS主要由NNO产生，主要是在培养的RGC中刺激的谷氨酸刺激的谷氨酸谷氨酸导致视网膜神经节细胞对视网膜神经节细胞的死亡，以浓度依赖于浓度依赖于浓度的方式。谷氨酸以浓度依赖性方式中细胞内Ca^<2+>浓度（[Ca^<2+>] _ I）增加。 NMDA和KCL引起相同的结果。 betaxolol抑制了[Ca^<2+>] _ I至25％的KCl诱导的增加。另一方面，贝物莫尔显着抑制谷氨酸诱导的RGC死亡。 [Ca^<2+>] _ I的增加被认为是谷氨酸神经毒性的第一个信号。 Betaxolol通过抑制电压依赖性钙通道产生神经保护作用。该方法可能可用于搜索神经保护药和新的青光眼治疗。较少的