Nogo-66蛋白对慢性高眼压大鼠视网膜小胶质细胞功能的影响

Effect of Nogo-66 on retinal microglia in chronic ocular hypertension rats

目的 探讨Nogo-66蛋白对慢性高眼压大鼠视网膜小胶质细胞表面抗原CD11b及MHC-Ⅱ表达的影响及其对损伤组织抗原呈递作用的干预性质.方法 对照实验研究.采用随机数字表法,将实验动物分为4组：高眼压＋Nogo-66蛋白组,正常眼压＋Nogo-66蛋白对照组,高眼压＋损伤对照组,正常眼压＋损伤对照组.应用532-激光行周边前房角光凝＋巩膜浅层静脉关闭术,建立慢性高眼压动物模型.实验组大鼠接种：于双后足趾及背部皮下多点注射无菌等渗10%Nogo-66蛋白磷酸缓冲液（PBS）,注射后7 d、1个月,分别以0.005%的Nogo-66蛋白PBS注射液2 μl进行玻璃体腔内注射.对照组大鼠接种：以等渗PBS等量于相同部位注射.初次接种后1个月,免疫组织化学法标记视网膜小胶质细胞表面抗原CD11b和MHC-Ⅱ,应用Image-Pro Plus Version 6.0图像分析软件进行图像处理,获得小胶质细胞表面抗原阳性表达的量化指标.实验组与对照组大鼠眼压均值比较,采用两样本计量资料的方差分析;小胶质细胞表面抗原阳性表达水平比较,采用配伍设计多个样本均数比较的t检验.结果 实验组大鼠于建模后7 d眼压开始升高,1个月时眼压最高为（24.16±2.70）mm Hg（1 mm Hg=0.133 kPa）,与对照组（15.93±3.28）mm Hg比较,差异有统计学意义（F=2.10,P〈0.05）.在Nogo-66蛋白干预下,正常眼压＋Nogo-66蛋白组大鼠视网膜小胶质细胞MHC-Ⅱ阳性表达百分比为（11.45±1.97）%,显著高于高眼压＋Nogo-66蛋白组（3.92±1.03）%,差异有统计学意义（t=2.14,P〈0.05）;正常眼压＋Nogo-66蛋白组大鼠视网膜神经节细胞层小胶质细胞CD11b表达百分比为（8.15±1.97）%,显著高于高眼压组（1.78±0.63）%,差异有统计学意义（t=2.35,P〈0.05）.结论 外源性Nogo-66蛋白能够刺激视网膜神经节细胞层及神经纤维层小胶质细胞表面抗原CD11b及抗原呈递分子MHC-Ⅱ的阳性表达,提示Nogo-66蛋白具有中枢免疫原性,其有可能激活抗原呈递细胞,完成其呈递损伤抗原的作用.

Objective To study the effect of protein Nogo-66 on the expression of CD11b and MHC-Ⅱ in retinal microglia of chronic ocular hypertension SD rats and the effects of protein Nogo-66 on the immunogenicity of injured retinal tissues.Methods It was a control experimental study.Chronic ocular hypertension rat model was established by laser photocoagulation on the anterior chamber angle and superficial vein of the sclera.One ml of Nogo-66（0.01%）in PBS was injected subeutaneouly on the day of laser treatment and 0.005% Nogo-66 PBS solution was injected into the vitreous 7days and 1 month latter.PBS without Nogo-66 was injected in the control group.The expression of cell surface antigen CD11b and MHC-Ⅱ were detected by immunohistochemistry 1 month and 1 day after the establishment of hypertension model.The difference of average IOP among groups was analyzed by variance analysis.The difierence of expression of CD11b and MHC-Ⅱ between the experimental and control groups was analyzed by t-test.Results The intraocular pressure （IOP） of experimental groups rised from the soventh day after modelbuilding and the highest IOP was（24.16±2.70）mm Hg （1 mm Hg=0. 133 kPa） 1 month later while that in the control groups was（15.93 ± 3.28 ）mm Hg. The difference between them was statistically significant （F= 2.10, P〈0. 05）. Expression of CD11b was （ 1.78 ± 0. 63 ）% and MHC-Ⅱ was （3.92 ± 1.03 ） % inNogo-66 with hypertension groups, these results was significantly lower than those in Nogo-66 with normal intraocular pressure groups in which the expression of CD11b was （ 8.15 ± 1.97 ） % （ t = 2.35, P 〈 0. 05 ）and MHC-Ⅱ was（11.45 ±1.97）% （t =2.14, P 〈0.05）. Conclusions Protein Nogo-66 activated the cell surface antigen CD11b in nerve fiber layer of retina and induced antigen presenting molecules （MHC-Ⅱ）. This indicates that Nogo has the center immunogenicity and this protein could activate antigenpresenting cells to present injury antigen.