雙七他克林對大鼠視網(wǎng)膜神經(jīng)節(jié)細胞興奮性毒素損傷的保護作用及機制研究
發(fā)布時間：2018-09-06 08:33
【摘要】： 目的：谷氨酸介導的興奮性毒性,主要與NMDA受體有關,是青光眼及其他多種視網(wǎng)膜疾病中視網(wǎng)膜節(jié)細胞死亡的重要原因。雙七他克林是一種非競爭性NMDA受體抑制劑,能夠減輕谷氨酸誘導的海馬神經(jīng)元損傷。在本實驗中,我們探討雙七他克林對NMDA誘導的視網(wǎng)膜節(jié)細胞膜電流的作用,以及對興奮性毒素所致視網(wǎng)膜節(jié)細胞損傷的保護作用。 方法：取出生1-3天SD乳鼠的視網(wǎng)膜,經(jīng)過兩步免疫法后,獲得純化的視網(wǎng)膜節(jié)細胞。培養(yǎng)7天后,使用膜片鉗記錄技術記錄雙七他克林對NMDA誘發(fā)的視網(wǎng)膜電流的作用。視網(wǎng)膜節(jié)細胞培養(yǎng)液中加入谷氨酸,再加入雙七他克林(0.01-1μM)或美金剛胺(1-10μM),培養(yǎng)6-24小時。使用MTT法測定細胞活力,Annexin V-FITC/PI法測定細胞凋亡。在動物實驗中,成年SD大鼠玻璃體注射谷氨酸(5μl,20nmol)或NMDA(5μl,40nmol),造成視網(wǎng)膜節(jié)細胞損傷。在注射谷氨酸或NMDA前15分鐘,腹腔注射雙七他克林(0.05,0.1,0.2mg/kg)或美金剛胺(20mg/kg)。使用HE染色,TUNEL染色和熒光金逆行標記分析視網(wǎng)膜節(jié)細胞損傷情況。 結果：全細胞膜片鉗記錄技術發(fā)現(xiàn),30μM的NMDA引起大約-50pA膜內(nèi)向電流,1μM可阻滯此電流。MTT實驗顯示,谷氨酸對體外培養(yǎng)的新生大鼠視網(wǎng)膜節(jié)細胞具有濃度和時間依賴性的損傷作用。雙七他克林和美金剛胺可阻止谷氨酸誘導的視網(wǎng)膜節(jié)細胞死亡,呈濃度依賴性,其IC50值分別為0.028μM和0.834μM。使用annexin V-FITC/PI染色法,證實了雙七他克林的抗凋亡作用。在體內(nèi)實驗中,TUNEL和逆行標記技術發(fā)現(xiàn),雙七他克林(0.2mg/kg)對谷氨酸誘導的視網(wǎng)膜節(jié)細胞有明顯的保護作用。HE染色和逆行標記結果顯示,玻璃體注射NMDA7天后,雙七他克林具有明顯的保護作用。TUNEL染色顯示在玻璃體注射NMDA18小時后,雙七他克林可有效減少節(jié)細胞層的凋亡細胞。 結論：我們的結果顯示,雙七他克林對體內(nèi)外興奮性毒素誘導的視網(wǎng)膜節(jié)細胞損傷均有保護作用,這種保護作用可能與抑制NMDA受體有關。這些發(fā)現(xiàn)使雙七他克林成為治療包括青光眼在內(nèi)的缺血性或創(chuàng)傷性視網(wǎng)膜病變的潛在藥物。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R774.1
本文編號：2225797
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R774.1
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