双七他克林对大鼠视网膜神经节细胞兴奋性毒素损伤的保护作用及机制研究

发布时间：2018-09-06 08:33

【摘要】： 目的：谷氨酸介导的兴奋性毒性,主要与NMDA受体有关,是青光眼及其他多种视网膜疾病中视网膜节细胞死亡的重要原因。双七他克林是一种非竞争性NMDA受体抑制剂,能够减轻谷氨酸诱导的海马神经元损伤。在本实验中,我们探讨双七他克林对NMDA诱导的视网膜节细胞膜电流的作用,以及对兴奋性毒素所致视网膜节细胞损伤的保护作用。方法：取出生1-3天SD乳鼠的视网膜,经过两步免疫法后,获得纯化的视网膜节细胞。培养7天后,使用膜片钳记录技术记录双七他克林对NMDA诱发的视网膜电流的作用。视网膜节细胞培养液中加入谷氨酸,再加入双七他克林(0.01-1μM)或美金刚胺(1-10μM),培养6-24小时。使用MTT法测定细胞活力,Annexin V-FITC/PI法测定细胞凋亡。在动物实验中,成年SD大鼠玻璃体注射谷氨酸(5μl,20nmol)或NMDA(5μl,40nmol),造成视网膜节细胞损伤。在注射谷氨酸或NMDA前15分钟,腹腔注射双七他克林(0.05,0.1,0.2mg/kg)或美金刚胺(20mg/kg)。使用HE染色,TUNEL染色和荧光金逆行标记分析视网膜节细胞损伤情况。结果：全细胞膜片钳记录技术发现,30μM的NMDA引起大约-50pA膜内向电流,1μM可阻滞此电流。MTT实验显示,谷氨酸对体外培养的新生大鼠视网膜节细胞具有浓度和时间依赖性的损伤作用。双七他克林和美金刚胺可阻止谷氨酸诱导的视网膜节细胞死亡,呈浓度依赖性,其IC50值分别为0.028μM和0.834μM。使用annexin V-FITC/PI染色法,证实了双七他克林的抗凋亡作用。在体内实验中,TUNEL和逆行标记技术发现,双七他克林(0.2mg/kg)对谷氨酸诱导的视网膜节细胞有明显的保护作用。HE染色和逆行标记结果显示,玻璃体注射NMDA7天后,双七他克林具有明显的保护作用。TUNEL染色显示在玻璃体注射NMDA18小时后,双七他克林可有效减少节细胞层的凋亡细胞。结论：我们的结果显示,双七他克林对体内外兴奋性毒素诱导的视网膜节细胞损伤均有保护作用,这种保护作用可能与抑制NMDA受体有关。这些发现使双七他克林成为治疗包括青光眼在内的缺血性或创伤性视网膜病变的潜在药物。
[Abstract]:Aim: glutamate mediated excitotoxicity, mainly associated with NMDA receptor, is an important cause of retinal ganglion cell death in glaucoma and many other retinal diseases. Gemcitrine is a non-competitive NMDA receptor inhibitor that can attenuate glutamate-induced hippocampal neuronal damage. In this study, we investigated the effects of dichlorcitrine on the membrane current of retinal ganglion cells induced by NMDA and the protective effects on the injury of retinal ganglion cells induced by excitatory toxin (excitotoxin). Methods: the retinal ganglion cells were obtained from 1-3-day SD rats by two-step immunoassay. After 7 days of culture, patch-clamp recording technique was used to record the effects of dichlortacrine on the retinal currents induced by NMDA. Glutamate was added to the culture medium of retinal ganglion cells and then was cultured for 6-24 hours with the addition of dimetacrine (0.01-1 渭 M) or amantadine (1-10 渭 M),) for 6-24 hours. Apoptosis was measured by MTT assay and Annexin V-FITC/PI assay. In animal experiment, vitreous injection of glutamic acid (5 渭 l) or NMDA (40 nmol) induced retinal ganglion cell injury in adult SD rats. At 15 minutes before injection of glutamate or NMDA, dimetacrine (0.05g / kg) or amantadine (20mg/kg) was injected intraperitoneally. The damage of retinal ganglion cells was analyzed by HE staining and fluorescent gold retrograde labeling. Results: the whole-cell patch clamp recording technique showed that about -50pA membrane current (1 渭 M) induced by 30 渭 M NMDA could block the current. The results showed that glutamate had a concentration-and time-dependent damage to cultured neonatal rat retinal ganglion cells (RGCs) in a concentration-and time-dependent manner. The concentrations of IC50 were 0.028 渭 M and 0.834 渭 M, respectively, in a concentration-dependent manner, and the IC50 values were 0.028 渭 M and 0.834 渭 M, respectively. The anti-apoptotic effect of dichlorcitrine was confirmed by annexin V-FITC/PI staining. In vivo, Tunel and retrograde labeling techniques showed that 0.2mg/kg had a significant protective effect on glutamic acid-induced retinal ganglion cells. The results of Tunel staining showed that the apoptotic cells in the ganglion layer could be effectively reduced by dimetacrine at NMDA18 hour after vitreous injection. Conclusion: our results suggest that dichlorcitrine has protective effects on excitotoxin induced retinal ganglion cell injury in vivo and in vitro, which may be related to the inhibition of NMDA receptor. These findings make Gemcitrine a potential drug for ischemic or traumatic retinopathy, including glaucoma.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R774.1

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