糖尿病大鼠视网膜神经细胞损伤机制的研究

The mechanism study of retinal nerve cell injury in rats with diabetes mellitus

目的观察糖尿病大鼠视网膜Müller细胞谷氨酸转运体(GLAST)、谷氨酰胺合成酶(GS)及胶质纤维酸性蛋白(GFAP)表达的变化、视网膜神经细胞凋亡的检测以及神经营养因子NT-3的表达,探讨糖尿病(DM)对视网膜神经细胞损伤的机制。设计实验研究。研究对象Sprague-Dawley(SD)大鼠82只。方法大鼠随机分为正常对照组12只,糖尿病模型组70只。链脲佐菌素诱导实验性DM大鼠模型。RT-PCR法检测视网膜GFAP mRNA表达水平;TUNEL法检测视网膜神经节细胞(RGC)及内核层细胞的凋亡并计数凋亡细胞数量;免疫组织化学技术LSAB法检测GFAP、GLAST、GS、NT-3在视网膜的表达,观察DM大鼠视网膜RGC及内核层细胞功能的改变,用图像分析仪测量免疫组化的显色强度。主要指标GFAP、GLAST、GS和NT-3的表达量,视网膜内核层和RGC细胞凋亡数。结果 (1)与正常组(1.00±0.02)相比,模型组GFAP阳性表达量(5.22±1.34)明显增加(P=0.000),GLAST、GS、NT-3阳性表达明显降低。(2)大鼠视网膜凋亡阳性细胞仅见于RGC层和内核层,模型组视网膜内核层细胞及RGC凋亡数量(36.00±6.02,11.48±2.08)比正常组(16.33±2.34,5.34±0.52)显著增加(P均=0.000)。(3)DM大鼠视网膜GFAP mRNA表达(7.00±0.37)比正常组(0.29±0.08)明显增加(P=0.000)。(4)GFAP阳性表达与内核层细胞及RGC凋亡数呈正相关(r=0.88、0.85,P=0.021、0.028);GLAST阳性表达与内核层细胞及RGC凋亡数呈负相关(r=-0.91、-0.89,P=0.014、0.020),GS阳性表达与内核层细胞及RGC凋亡数呈负相关(r=-0.93、-0.90,P=0.007、0.009);NT-3阳性表达与内核层细胞及RGC凋亡数呈负相关(r=-0.74、-0.71,P=0.036、0.041)。结论糖尿病大鼠视网膜神经细胞凋亡增加与Müller细胞的过度反应性增生及神经营养因子的缺失有关,高浓度谷氨酸的兴奋性毒性作用以及神经营养因子NT-3的缺失是其视网膜神经细胞损伤的重要机制。

Objective To assess the expression of GFAP,GLAST,GS and NT-3 in Müller cells and the apoptosis of retinal nerve cells in rats with diabetes mellitus（DM） and to discuss the mechanisms of retinal nerve cell injury in DM rats.Design Experimental study.Paticipants 82 Sprague-Dawley（SD） rats.Methods Diabetic model in rats was induced by streptozotocin method.The rats are randomly divided into 2 groups： model group and normal group.The expression of GFAPmRNA in retina was measured by RT-PCR.LSAB method was used to measured the expression level of GFAP,GLAST,GS,NT-3 in retinal Müller cells.Apoptosis of retinal nerve cells was measured by TUNEL assay and apoptosis cells were counted.Immunological histochemistry assay was used for quantitative analysis.Main Outcome Measures The expression of GFAP,CLAST,GS,and NT-3;the number of apoptotic cells in inner nuclear layer and RGC layer.Results Compared with normal group（1.00±0.02）,the positive expression of GFAP in the retina of diabetic rats（5.22±1.34） was increased significantly（P=0.000）.The positive apoptotic retinal cells were only expressed in RGCs and inner nuclear layer.The numbers of apoptotic cells in RGCs and inner nuclear layer of diabetic rats（36.00±6.02 and 11.48±2.08） were significantly higher than that of normal group（16.33±2.34 and 5.34±0.52）（all P=0.000）.Compared with normal group（0.29±0.08）,the expression of GFAPmRNA in the retina of diabetic rats（7.00±0.37） was enhanced significantly（P=0.000）.The number of apoptotic RGCs and cells in inner nuclear layer was correlated positively with the positive expression level of GFAP（r=0.88,0.85,P=0.021,0.028）,and was negatively correlated with the expression of GLAST,GS and NT-3（r=-0.74^-0.93,-0.71^-0.90;all P0.05）.Conclusion The number of apoptosis of retinal nerve cell is closely related to the active proliferation of glial reactive state in retinal Müller cells and the deficiency of neurotrophic factors.The excitotoxicity of high concentrations of glutamate and the deficiency of neurotrophic factors are important mechanism of retinal nerve cell injury in diabetes.