【作者】 许红霞；

【导师】 糜漫天；

【作者基本信息】 第三军医大学 ， 营养与食品卫生学， 2004， 博士

【摘要】 谷氨酸（glutamate,Glu）是中枢神经系统主要的神经递质，它介导兴奋性突触传递。但突触间过量的Glu会引起神经元损伤甚至死亡，称为Glu的兴奋毒性。大量实验证据显示，Glu的兴奋毒性在中枢神经系统缺血、创伤及神经退行性疾病的神经元死亡中起着重要作用。现已明确，Glu的兴奋毒性是Glu在突触间的过量释放及对突触后神经元的进一步激活。其作用机制为：过量释放的Glu激活突触后膜上大量的谷氨酸受体，引起相应的离子通道开放，影响Na⁺、Cl^-、Ca²⁺等的跨膜流动，主要导致细胞内Ca²⁺浓度升高(称为细胞内Ca²⁺超载)，过量的Ca²⁺激活细胞内Ca²⁺依赖的信号级联途径，最终导致神经细胞死亡。其中，细胞内Ca²⁺超载是Glu兴奋毒性引起神经细胞死亡的中心环节。Glu增加细胞内Ca²⁺主要是通过以下几种机制：①激活可透过Ca²⁺的离子型NMDA受体通道：②激活离子型AMPA受体引起膜去极化从而开放电压依赖的Ca²⁺通道；③激活代谢型谷氨酸受体导致其耦联的磷酸肌醇水解，后者使Ca²⁺从细胞内钙池释放。 Glu亦是视网膜中最主要的兴奋性神经递质，感光细胞和双极细胞均以Glu为递质，视网膜神经节细胞（Retinal ganglion ceils,RGCs）接受双极细胞的Glu能突触传递。很早就观察到兴奋性氨基酸对视网膜内层细胞的毒性作用，其中RGCs对Glu的兴奋毒性最为敏感。目前认为Glu兴奋毒性是视网膜缺血—再灌注损伤、缺氧、青光眼、视网膜变性性疾病等的RGCs死亡的主要机制之一。研究表明：RGCs及视网膜其它内层神经元的兴奋毒性损伤主要是由对NMDA受体的过度刺激引起的。AMPA及KA受体也参与Glu对RGCs的损伤，Glu能激活培养RGCs的AMPA／KA受体，引起细胞内Ca²⁺浓度增加，降低RGCs存活。Glu介导的神经细胞死亡中，既有急性的细胞坏死，又有迟发性的细胞凋亡存在。大量研究表明，预防性阻断Glu兴奋毒性可以保护RGCs，减少视网膜损伤和RGCs死亡。目前预防Glu兴奋毒性保护RGCs的研究主要集中于两方面：（1）拮抗NMDA受体、AMPA及KA受体；（2）降低细胞内Ca²⁺超载，如Ca²⁺通道阻断剂。但由于NMDA受体在体内同时介导其它正常的生理作用，将其受体拮抗剂用于人体会对中枢神经系统产生副作用，使其临床应用受到限制。对第三军医大学博士学位论文Ca2+通道阻断剂的临床应用研究也刚刚起步。 牛磺酸（t aurine）是一种日丙氨酸，它是中枢神经系统最丰富的游离氨基酸之一，在许多哺乳动物的大脑中其浓度甚至超过了Glu。牛磺酸在中枢神经系统可作为渗透压调节剂和抑制性神经调质。早就发现，牛磺酸能减轻癫痛病的症状，近来发现，牛磺酸还具有神经保护作用，这可能与其对抗Glu及其类似物诱发的兴奋毒作用有关。目前公认，牛磺酸是一个调节及降低神经细胞内ca2+水平的强有力因子。有研究提示，牛磺酸的神经保护机制与其防止或降低Glu诱导的胞内CaZ十水平的升高有关。牛磺酸在视网膜也有广泛的生理功能，它是视网膜发育所必需的营养因子，具有调节渗透压、抗氧化、调节ca2+、调节蛋白磷酸化等作用，但目前未见牛磺酸用于防护视网膜尤其是RGCs的Glu兴奋毒性的研究。 基于以上分析，结合国内外研究进展，本课题通过体内和体外研究观察牛磺酸对Glu引起的RGCs兴奋毒性的防护作用。体内研究通过在大鼠玻璃体内注射Glu造成视网膜Glu兴奋毒性的动物模型，通过视网膜全铺片RGCs计数、视网膜电图（E lectroretinograms，ERG）、视网膜组织结构及超微结构观察等方法观察牛磺酸对大鼠视网膜Glu兴奋毒性的体内防护作用;并运用原位TUNEL检测、免疫组化、免疫印迹、RT一PCR等技术，进一步观察牛磺酸对Glu所致的视网膜RGCs凋亡、视网膜Glu、Thy一1、神经丝蛋白轻链（neurofilaments light ehain，NF一L）、神经胶质原纤维酸性蛋白（olial fibrillary aeid protein，G队P）蛋白及mRN^的表达水平改变的影响。体外研究采用两种细胞模型:原代培养的大鼠视网膜神经元及原代纯化培养的大鼠RGCs，分别以终浓度为1幻以们比oUL的Glu作用于原代培养视网膜神经细胞10而n、25 p moUL Glu作用于RGCs3天制备Glu兴奋毒性模型。通过台盼蓝拒染实验、钙黄绿素一AM染色存活细胞、TUNEL检测、Thy一l蛋白表达检测等技术观察牛磺酸对培养的视网膜神经细胞、纯化培养的RGcs Glu兴奋毒性的防护效应;利用激光共聚焦ca2+成像技术，观察牛磺酸对Glu引起的培养Rocs细胞内ca2+（[ ca2+li）超载的影响，并通过对Glu及牛磺酸影响RGcs[C a2+li的动态观察，从牛磺酸调节Glu兴奋毒性所致细胞内ca2+失衡的角度，探讨牛磺酸防护大鼠RGCs Glu兴奋毒性的机制。 本研究主要实验结果如下: 1.视网膜全铺片计数大鼠视网膜节细胞层（Retinal ganglion eells，GCL）细胞，发现:正常的视网膜，GcL的平均细胞密度是6426个/毫米2，其中36%是RGcs，64%是非RGCs细胞。玻璃体内注射Glu使GCL的细胞数显著减少，以RGCs的减少为主（减少75%），腹腔注射高剂量（25m叭g体重）牛磺酸使RGCs丢失显著减少，而低剂量第三军医人学博士学位论文（5m岁kg）牛磺酸的作用不明显。玻璃体注射Glu使视网膜内网状层(Inner plexiform更多还原

【Abstract】 As the major neurotransmitter in mammalian central nervous system (CNS), Glutamate (Glu) contributes to excitatory neurotransmission in many central synapses. However, extracellular glutamate in excess can lead to neuronal injury or death, this so-called glutamate-induced excitotoxicity has been implicated as an important mechanism underlying a variety of brain injuries such as ischemia, trauma,and neurodegenerative diseases of CNS. The key reason for this excitotoxicity is because glutamate can increase the concentration of intracellular Ca2+ ([Ca2+];) in neurons, and thus finally induce neuronal death by activating proteases, phospholipases, and endonucleases. Several mechanisms explain how glutamate increases intracellular Ca2+ level. The activation of Ca2+-permeable N-methyl-D-aspartate (NMDA) receptors, opening of voltage-dependent Ca2+ channels following membrane depolarization induced by activation of 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and/or activation of metabotropic glutamate receptors(mGluRs) linked to phosphoinositide hydrolysis, which releases Ca2+ from intracellular stores are leading factors.Glutamate is the principal excitatory neurotransmitter in retina as well as in CNS, and retinal ganglion cells (RGCs) are primarily glutamatergic. Glutamate-induced excitotoxicity has been implicated as an important cause of death of RGCs in retinal ischemia, glaucoma, and degenerative retinopathy. Over-stimulating NMDA receptors contributes to the death of glutamate-induced inner retinal neurons include RGCs. Some researches indicate that low concentration glutamate can activate Ca2+-permeable AMPA-KA receptors in cultured RGCs, leading to increases in [Ca2+], and decreased survival, suggesting that the activation of AMPA-KA receptors may also play a role in neurotoxicity in retinal neurons. Accumulating evidence suggests that blocking glutamate excitotoxicity can protect RGCs and reduce RGCs death, which is the theoretical foundation of the current two methods that are provided to protect RGCs from glutamate-induced excitotoxicity: (1) Glutamate -receptor antagonists. However, since NMDA-receptor activities are essential for normal neuronal function, potential neuroprotective agents that block virtually all NMDA-receptoractivity will have unacceptable clinical side effects. (2) Agents to reduce Ca2+ overload, such as Ca2+-channel blocker. It is just the beginning of these kinds of research.Taurine, another one of the most abundant free amino acid in CNS other than glutamate, is a 0 -amino acid. As a matter of fact, the concentration of taurine in the brain even exceeds that of glutamate in many mammals. Taurine is known to act as an osmoregulator and an inhibitory neuromodulator in CNS. It relieves epileptic seizures and serves as a neuroprotector, which may explain its protective effect against glutamate-induced excitotoxicity. There is a consensus that taurine is a powerful agent in regulating and reducing the intracellular calcium levels in neurons, it functions in retina as osmoregulatory agent, anti-oxidant, and Ca2* modulator. In short, taurine is an essential nutrient agent in retinal development, but the effects of taurine on glutamate-induced excitotoxicity in RGCs are still poorly understood.Based on the researches from both at home and abroad, the purpose of this study was to determine whether taurine has neuroprotective effects in experimental in vitro and in vivo models. During in vivo study, we used a rat model of retinal glutamate excitotoxicity induced by intravitreal injection of 40nmol glutamate. The retinal damage was evaluated by counting the number of cells in the ganglion cell layer (GCL), examining the a- and b-waves in the electroretinogram (ERG), Hematoxylin-eosin (HE) staining, and observing ultrastructure of retina. For the purpose of advanced research in the protective effects of taurine, situ TUNEL test, immunohistochemistry, western blotting, and RT-PCR methods were used to study the RGCs apoptosis, the expression of glutamate, Thy-1, Neuro更多还原