目的 探讨玻璃体腔注射神经生长因子(NGF)对早期糖尿病大鼠视网膜神经节细胞(RGC)的保护作用及其可能机制.方法 取SD大鼠按照随机数字表法分为正常对照组、糖尿病模型组、磷酸盐缓冲液(PBS)组和NGF组,每组各6只,其中糖尿病模型组、PBS组和NGF组大鼠用链脲佐菌素(STZ)腹腔内注射建立糖尿病大鼠动物模型,正常对照组大鼠常规培养.PBS组及NGF组建模成功后4周玻璃体腔分别注射PBS和NGF(0.5μg/μl)各2μl,均取右眼为实验眼,每周注射1次,连续4周.给药4周后使用Phoenix MicronⅣ小动物视网膜成像系统观察各组大鼠视网膜微血管病变,各组过量麻醉法处死1只大鼠眼球制备超薄切片,透射电子显微镜下观察;取5只大鼠眼球制备视网膜石蜡切片,TUNEL法观察RGC凋亡指数,免疫组织化学法分析RGC bcl-2蛋白及bax蛋白的表达.结果 糖尿病模型大鼠体质量较正常对照组明显减轻,进食进水及尿量明显增多.各组大鼠造模后4周体质量和血糖值比较,差异均有统计学意义(F=202.352、148.444,均P＜0.001).早期糖尿病大鼠眼底照相显示,未见明显糖尿病视网膜微血管病变,透射电子显微镜下可见糖尿病模型组及PBS组较正常对照组RGC数目明显减少,细胞膜皱缩,细胞质浓缩,细胞器水肿,染色质边集明显,凋亡增加,NGF组RGC损害较糖尿病模型组及PBS组轻;正常对照组、糖尿病模型组、PBS组和NGF组的凋亡指数分别为(3.88±1.28)％、(92.56±1.58)％、(92.64±2.30)％和(59.34±3.89)％,各组凋亡指数总体比较差异有统计学意义(F=854.554,P＜0.001);糖尿病模型组RGC凋亡指数较正常对照组明显增加,NGF组RGC凋亡指数较糖尿病模型组及PBS组明显减少,差异均有统计学意义(均P＜0.05);正常对照组、糖尿病模型组、PBS组和NGF组的bcl-2蛋白表达量分别为38.11±1.01、22.38±3.90、23.04±3.14和84.69± 1.45,总体比较差异有统计学意义(F=366.206,P＜0.001).正常对照组、糖尿病模型组、PBS组和NGF组的bax蛋白表达量分别为4.22±1.89、56.59±6.67、56.30±8.51和26.19±2.44,总体比较差异有统计学意义(F=61.435,P＜0.001).糖尿病模型组较正常对照组bcl-2蛋白表达明显减少,bax蛋白表达明显增加,差异均有统计学意义(均P＜0.05).NGF组bcl-2蛋白表达较PBS组及糖尿病模型组明显升高,bax蛋白表达明显下降,差异均有统计学意义(均P＜0.05). 结论 早期糖尿病大鼠在眼底照相未发现糖尿病视网膜微血管病变时就已经发生RGC超微结构的损害.玻璃体腔注射NGF可能通过抑制糖尿病模型大鼠RGC的凋亡对视网膜神经元发挥保护作用.%Objective To investigate the protective effect of intravitreal injection of nerve growth factor (NGF) on apoptosis of retinal ganglion cells (RGCs) in early diabetic rats and its possible mechanism.Methods SD rats were divided into normal control group,diabetic model group,phosphate buffer solution (PBS) group and NGF group according to random number table method,with each group contain 6 rats.The rats in diabetic model group,PBS group and NGF group were injected intraperitoneally with streptozotocin (STZ) to establish diabetic rat model.After 4 weeks of modeling,2 μl PBS and 2 μl NGF (0.5 μg/μl) were injected into the vitreous cavity in PBS group and NGF group,respectively.The right eyes served as the experimental eyes,inject once a week for 4 weeks.After 4 weeks of injection,the retina microangiopathy of each group was observed by Phoenix Micron Ⅳ small animal retinal imaging system.After high dose anesthesia,the eyeball of one rat from each group was taken to prepare ultrathin sections and observed by transmission electron microscopy.Five rat eyeballs were taken to prepare retinal paraffin sections from each groups.The RGC apoptotic index was observed by TUNEL method.The expressions of RGC bcl-2 protein and bax protein were analyzed by immunohistochemistry.This study was approved by the Experimental Animal Ethics Committee of Central South University,and the experimental procedures were in accordance with the National Institutes of Health(NIH) guidelines for the Care and Use of Laboratory,and follow the 3R principle.Results The weight of the diabetic model rats was significantly lower than that of the normal group,and the intake of water and food were significantly increased,the urine volume was also in creased.The body weight and blood glucose level of the rats were significantly different among different groups after 4 weeks of modeling (F=202.352,148.444,both at P＜0.001).Fundus photography of early diabetic rats showed no obvious diabetic retinal microangiopathy.The numbers of RGC in the diabetic model group and PBS group were significantly lower than that in the normal control group under the transmission electron microscope.The membrane shrinkage,cytoplasmic condensation,organelle edema,chromatin peripheral collection were obvious and the cell apoptosis number was increased.The RGC lesions in the NGF group were lighter than those in the diabetic model group and PBS group.The apoptotic indexs in the normal control group,diabetic model group,PBS group and NGF group were (3.88±1.28)％,(92.56±1.58)％,(92.64±2.30)％ and (59.34±3.89)％,respectively,the overall difference of apoptotic index between the four groups was statistically significant (F=854.554,P＜0.001);the RGC apoptotic index of the diabetic model group was significantly higher than that of the normal control group.The RGC apoptotic index of the NGF group was significantly lower than that of the diabetic model group and PBS group (both at P＜0.05).The expression levels of bcl-2 protein in normal control group,diabetic model group,PBS group and NGF group were 38.11 ± 1.01,22.38±3.90,23.04±3.14 and 84.69±1.45,respectively,with a significant difference among the groups (F =366.206,P＜0.001).The expression levels of bax protein in normal control group,diabetes model group,PBS group and NGF group were 4.22± 1.89,56.59±6.67,56.30±8.51 and 26.19±2.44,respectively,with a significant difference among the groups (F=61.435,P＜0.001).The expression of bcl-2 protein in the diabetic model group was significantly lower than that in the normal control group (P＜0.05),and the expression of bax protein was significantly increased (P＜0.05).The expression of bcl-2 protein in NGF group was significantly higher and the expression of bax protein was significantly lower than those in the PBS group and diabetic model group (all at P＜0.05).Conclusions The ultrastructural damage of RGCs has occurred in early diabetic rats without diabetic retinal microangiopathy.Intravitreal injection of NGF may produce retinal neuroprotective effects by inhibiting apoptosis of RGCs in diabetic rats.
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