葡萄糖调节蛋白在急性高眼压大鼠视网膜中的表达及其意义

背景 青光眼导致的视网膜神经节细胞(RGCs)进行性死亡是导致患者视功能损害的主要病理基础,研究表明内质网应激(ERS)参与此过程,葡萄糖调节蛋白78( GRP78)是内质网中的特异性标志物,检测GRP78在高眼压诱导视网膜中的表达对青光眼患者视神经功能保护机制的研究具有重要意义. 目的 检测GRP78在大鼠急性高眼压后不同时期视网膜中的表达情况,探讨ERS在急性青光眼损伤中的作用.方法 56只Wistar大鼠采用随机数字表法分为正常对照组、前房穿刺组、急性高眼压12h组及急性高眼压1、3、7、14 d组,每组8只眼.急性高眼压模型的制作采用前房穿刺灌注生理盐水法,眼压提高至66 mmHg.分别于造模后12h及1、3、7、14 d用过量麻醉法处死大鼠.苏木精-伊红染色法观察各组大鼠视网膜的病理形态学改变；免疫组织化学法检测视网膜中的GRP78蛋白表达的变化；实时定量逆转录聚合酶链反应(RTPCR)法检测视网膜中GRP78 mRNA的表达变化. 结果 正常对照组大鼠视网膜各层细胞排列整齐,急性高眼压后12h视网膜水肿增厚、细胞核肿胀,1d时视网膜形态学异常改变达到高峰,3d后水肿减轻,视网膜变薄.免疫组织化学染色显示正常对照组、前房穿刺组大鼠RGCs层和内核层GRP78蛋白呈弱阳性表达,A值分别为0.195±0.006、0.196±0.005,急性高眼压12h组大鼠GRP78在视网膜的表达明显增强,A值为0.293±0.011,造模后1d时GRP78表达强度达到高峰,A值为0.499±0.039,造模后3d时GRP78表达明显降低,A值为0.268±0.017,与正常对照组大鼠比较差异均有统计学意义(t=0.098、0.304、0.073,P＜0.05),但造模后7d和14 d时GRP78在大鼠视网膜RGCs层和内核层中的表达量接近正常,与正常对照组比较差异均无统计学意义( t=0.002、0.001,P＞0.05).实时定量RT-PCR检测表明,GRP78 mRNA在正常对照组大鼠视网膜的相对表达量( 2-△△CT)为1.011±0.013,眼压升高12 h快速上调,为1.536±0.145,1 d时达高峰,为2.141±0.171,3d时迅速降低,为1.420±0.212,与正常对照组比较差异均有统计学意义(t=0.525、1.130、0.409,P＜0.05),而造模后7d及14 d GRP78 mRNA在大鼠视网膜的表达量明显下降,与正常对照组比较差异均无统计学意义(t=0.020.0.004,P＞0.05).结论 GRP78参与大鼠急性高眼压后视网膜损伤的发生机制,提示通过对ERS过程进行干预可能达到保护急性高眼压眼视神经功能的目的.

Expression of glucose-regulated protein in rat retina with acute high intraocular pressure and its significance

Background The progressive death of retinal ganglion cells （RGCs） is the primary pathological characteristics of visual defects in glaucomatous eye. Research showed that endoplasmic reticulum stress （ ERS ） is involved in this progression. Glucose-regulated protein 78 （GRP78） is a special marker of ERS. To understand the change in expression of GRP78 in the retina under the pressure induced is very important for the protection of visual function. Objective The present study was to observe the expression of GRP78 in rat retina with acute high intraocular pressure （IOP） and investigate the possible effect of ERS in acute glaucoma damage. Methods Fifty- six Wistar rats were randomly assigned to normal control group, anterior chamber punctured group and 12 hours, 1 day,3,7,14 days groups following acute IOP rising. The acute high IOP models were established in the right eyes of 40 Wistar rats by paracentesis of the anterior chamber and infusion of normal saline solution into the anterior chamber. The histopathology changes of the retina were examined by hemotoxylin and eosin staining. The expression of GRP78 protein and mRNA in the retina were detected by immunohistochemistry and real-time fluorescence quantitative PCR. Results The retinal layers and ceils were clear with normal alignement in the rats of the normal control group and the anterior chamber punctured group. The edema and thickening of retinas appeared in 12 hours after molding and peaked in 1 day after molding. Then the retina decreased in thickness and atrophied from 3 days through 14 days. The expression levels of GRP78 protein （A value） were 0. 195 ± 0. 006 in the normal rats and gradually increased from 12 hours through 3 days （0. 268±0. 017） following molding with the peak value at 1 day （0. 499±-0. 039 ）, showing significant differences in comparison with the normal control group （t = 0. 098,0. 304, 0. 073 ,P〈0. 05）,and the reduction in expression at 7 and 14 days were not significantly different from the normal control group （t = 0. 002,0. 001, P〉0.05 ）. The relative value of GRP78 mRNA （2AAct） in the retina was 1. 011 _± 0. 013 in the normal control group and gradually up-regulated from 12 hours through 3 days with the peak value at 1 day （2. 141±0. 171 ） and then down-regulated from the third day onwards. A significant difference was seen in 2-△△CT value between the normal control group and 12 hours group, 1 day group or 3 group （t = 0. 525,1. 130,0.409, P〈0.05 ）. However,the 2 -△△CTvalues of GRP78 mRNA at 7 and 14 days was similar to that of the normal control group （t= 0. 020,0. 004, P〉0. 05 ）. Conclusions GRP78 participates in the process of RGCs damage following acute high IOP. The results suggest that interfering with ERS may be helpful for protecting the retina and optical nerve from pressure-induced damage.