Molecular biological and molecular genetic studies in apoptosis of retinal ganglion cells and retinal differentiation.

本研究观察了大鼠视网膜细胞（RGC）在生后发育不同阶段突触发生过程中一氧化氮合酶（NOS）亚型的表达及一氧化氮（NO）的合成。将来自产前、哺乳期、年轻和成年大鼠的reina固定在多聚甲醛中。取多聚甲醛固定的大鼠神经节细胞冰冻切片，用激光共聚焦显微镜观察NOS的组成型（n型和eNOS型）。体外谷氨酸刺激RGCs合成NO。用二氨基荧光素-2-二乙酸酯（diaminofluorescein-2 diacetate，a）荧光指示剂真实的检测细胞内NO水平，免疫组化法检测nNOS和eNOS的表达。在培养的RGCs细胞内NO水平在20分钟的观察过程中表现出自发波动。NOS的非特异性抑制剂L-NAME和nNOS的特异性抑制剂7-NI的存在显著抑制了L-精氨酸和精氨酸加入培养液后6和8 min细胞内NO的升高。本研究揭示了所有组成型NOS异构体都在RGCs中表达，并证明NO主要由培养的RGCs中的glitamete刺激的nNOS产生。我们致力于研究衰老是否会降低组织脑源性神经营养因子（BDNF）及其受体TrkB的表达水平，这被认为是支持RGCs的存活。

The expression of isoforms of nitric oxide sythase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal cells (RGCs) during synaptogenesis for variation phases of the pre- and postnatal developmental periods were investigated. The reinas from prenatal, lactaing, young, and adult rats were fixed in paraformaldehyde. The cryosections or paraformaldehyde-fixed ganglion cells purified from rat pupus were immunostained for constitutive isoforms of NOS(n and eNOS) and observed with confocal laser scanning microscope. Synthesis of No in the RGCs was achieved by in vitro stimulation with glutamete. The intracellular NO levels measured I real time using diaminofluorescein-2 diacetate, a, fluorescence indicator of NO. immunohistochemical analysis revaled nNOS and eNOSin vitro. Intracellular NO levels in cultured RGCs showed spontaneous fluctuation during a 20-min observation. The presence of both a non-specific NOS inhibitor, L-NAME, and a specific nNOS inhibitor, 7-NI, significantly inhibited the increase of intracellular NO 6 and 8 min after the introduction of L-arginine and glutamete to the medium. This study revealed that all constitutive NOS isoforms are expressed I RGCs and demonstrated that NO is produced by nNOS mainly through stimulation by glitamete in cultured RGCs. We addressed to examine if senescence may decrease the expression levels of tissue brain-derived neurotrophic factor (BDNF) and its receptor TrkB which are thought to support the survival of RGCs.