αA-晶体蛋白在2型糖尿病大鼠神经视网膜的异常表达

Abnormal expression of αA-crystallin in neural retina in type 2 diabetic rats

目的 用蛋白质组学方法观察αA-晶体蛋白在早期2型糖尿病大鼠神经视网膜的异常表达.方法 28只成年雄性Sprague-Dawley（SD）大鼠随机分为正常对照组和糖尿病组.每组14只.糖尿病组大鼠采用高脂饮食饲养联合小剂量链脲佐菌素（STZ）注射诱导2型糖尿病模型（T2DM）,以随机血糖持续高于16.7 mmol/L为模型建立成功标准.对照组用常规饲料喂养.腹腔注射相同剂量枸橼酸钠缓冲液.成模56 d后断颈处死所有大鼠,剥离留存神经视网膜组织.从对照组和糖尿病组各取3只大鼠的神经视网膜组织,提取总蛋白,用二维凝胶电泳（2-DE）方法分别进行分离.应用基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-MS/MS）和肽质量指纹图谱（PMF）技术对部分差异表达蛋白进行鉴定,并用蛋白免疫印迹（Western blot）和间接免疫荧光（IMF）方法对在糖尿病组表达上调的αA-晶体蛋白进行验证.结果 平均每块凝胶检测到的蛋白斑点,对照组为（3122±37）,糖尿病组为（2702±21）个.二者比较,表达水平差异有统计学意义的斑点约150个（t值均〉2.57,P值均〈0.05）.在糖尿病组表达上调68个,表达下调82个.质谱分析和数据库检索鉴定出20个差异表达蛋白斑点.其中二维凝胶中2369和1048斑点在糖尿病组呈现高表达.经质谱PMF鉴定为αA-晶体蛋白.Western blot检测结果显示,αA-晶体蛋白在糖尿病组大鼠神经视网膜表达明显增高.IMF结果显示αA-晶体蛋白在糖尿病组高表达,阳性表达主要位于视网膜的内外核层,在细胞的核周细胞浆区域有强荧光聚集.结论 αA-晶体蛋白在早期2型糖尿病大鼠神经视网膜表达增高.

Objective To observe the abnormal expression of aA-erystallin protein in neural retina in type 2 diabetic rats via proteomic technique. Methods Twenty-eight male Sprague-Dawley （SD） rats were randomly divided into the normal control and the diabetic experimental groups with 14 rats in each group. A type 2 diabetes rat model （T2DM） was set up in the diabetic experimental group by feeding high-fat diet combined with peritoneal injection of low-dose streptozotocin （STZ） the successful diabetes model is with the randomly-detected blood glucose of 〉16.7 mmol/L. The rats in the control group underwent peritoneal injection of equivalent sodium citrate solution and were fed with normal diet. All of the animals were sacrificed by decapitation 56 days after the induction of diabetes. The eyes were enucleated and the neural retina layers were carefully peeled off and preserved. The total neural retinal proteins were extracted from the control and diabetic groups, respectively, and then subjected to two-dimensional gel electrophoresis （2-DE）. Some different proteins spots were identified by peptide mass fingerprinting （PMF） as well as by tandem mass spectrometric （MS/MS） measurements. Western blot and indirect immunofluorescence （IMF） were used to confirmed that aA-crystaUin protein expression was upregulated in diabetic retina. Results An average of （3122 ± 37） spots in normal retinas and（2702 ±21）spots in diabetic group were found by 2-DE image analysis software about 150 spots in 2-DE gel of diabetic retinae exhibited statistically significant variations （t〉2.77, P〈0. 05）. Compared with normal rats＇ retinae, diabetic ones presented 68 protein spots of up-regulation expression and 82 of down-regulation expression in 2DE gel. Furthermore, 20 of the 150 protein spots were identified by mass spectrometry. The points of 2369 and 1048 in 2-DE gel, showing high expression in diabetic retinal tissues, were identified as aA-crystallin via PMF. Western blot validated that the expression level of αA-crystallin in diabetic neural retina was much higher than that in the control group. Significantly increased expression of αA-crystallin in nuclear retina in diabetic group was also observed by IMF. Fluorescence was mainly seen in the retinal nuclear layer αA-crystallin aggregation was detected in the perinuclear region of neurons. Conclusion The expression of αA-crystatlin increases in neural retina of early T2DM rats.