内皮黏蛋白对糖尿病大鼠视网膜神经元的保护作用及其机制  

Protection effect of endomucin on retinal neurons and its mechanism in streptozotocin-induced diabetic rat

作　　者：牛田 谢莉萍[1] 邢馨丹 蒋炎 金慧昳 王海燕[1] 刘堃[1] Niu Tian;Xie Liping;Xing Xindan;Jiang Yan;Jin Huiyi;Wang Haiyan;Liu Kun(Department of Ophthalmology,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,Chin)

机构地区：[1]上海交通大学附属第一人民医院眼科,200080

出　　处：《中华实验眼科杂志》2018年第6期417-423,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金面上项目（81470638）;上海市卫健委优秀学科带头人计划项目（2017BR056）;上海市教育委员会高峰高原学科建设计划项目（20161426）

摘　　要：目的 观察1型糖尿病大鼠视网膜组织内皮黏蛋白（EMCN）表达变化,探讨其通过活化蛋白激酶B（Akt）对1型糖尿病模型大鼠神经元的保护机制.方法 采用随机数字表法将健康清洁级SD大鼠分为正常对照组、糖尿病模型组、EMCN转染组和mCherry转染组4个组,每组14只,造模前2周玻璃体腔内注射生理盐水、腺相关病毒（AAV）、AAV-EMCN或AAV-mCherry,分别收集造模后4周和8周视网膜组织蛋白样本.采用Western blot法检测各组大鼠EMCN的表达;利用视网膜铺片观察AAV对造模后8周大鼠视网膜的转染效率;采用苏木精-伊红染色观察造模后8周各组大鼠视网膜组织形态结构变化;采用免疫荧光染色检测造模后8周各组大鼠视网膜组织细胞中cleaved caspase-3阳性细胞情况;采用TUNEL法观察各组大鼠造模后8周视网膜组织凋亡细胞情况;采用Western blot法检测造模后8周各组大鼠视网膜组织中Akt磷酸化比例. 结果糖尿病模型组、EMCN转染组和mCherry转染组大鼠空腹血糖（FPG）均较对应时间点正常对照组高,差异均有统计学意义（均P＜0.001）.糖尿病模型组大鼠造模后4周、8周视网膜组织中EMCN表达量均低于正常对照组,差异均有统计学意义（t=3.71,P＜0.05;t=10.09,P＜0.001）.造模后8周,mCherry转染组大鼠视网膜铺片间可见大量红色荧光,表明AAV转染视网膜细胞成功.糖尿病模型组大鼠视网膜组织中EMCN蛋白相对表达量较正常对照组显著下降,差异有统计学意义（t=13.67,P＜0.001）,EMCN转染组大鼠视网膜组织中EMCN表达量较糖尿病模型组增加,差异有统计学意义（t=3.18,P＜0.05）,mCherry转染组视网膜组织中EMCN表达水平与糖尿病模型组比较,差异无统计学意义（t=2.31,P=0.08）.正常对照组大鼠视网膜组织结构正常,糖尿病模型组及mCherry转染组大鼠视网膜组织内界膜肿胀,视网膜神经节细胞（RGCs）减少,内核层及外核层结构疏松排Objective To investigate the change of endomucin（EMCN） expression in diabetic retinopathy （DR） and its protective role in neurons apoptosis.Methods Fifty-six clean SD rats were randomly divided into 4 groups,including normal control group with intravitreal injection of normal saline,diabetes mellitus （DM） group with intravitreal injection of normal saline,EMCN transfection group with intravitreal injection of adenovirus associated virus（AAV）-EMCN and mCherry transfection group with intravitreal injection of AAV-mCherry,14 rats for each group.Intravitreal injection was performed 2 weeks before diabetes modeling.Western blot analysis was used to measure the expression of EMCN and phosphorylated Akt （p-Akt）/Akt.Flat-mounted retinas were performed to test the transfection efficiency.Hematoxylin-eosin staining was performed to examine the morphology of retinal tissue.The expression of cleaved caspase-3 in retinas of rats was assayed by immunofluorescence.The retinal apoptotic cells were detected by TUNEL.The use and care of the rats followed the ARVO Statement.Results The levels of fasting plasma glucose were significantly higher in the DM group,EMCN transfection group and mCherry transfection group than those in the normal control group （all at P＜0.001）.The expression of EMCN protein at 4 weeks and 8 weeks after modeling in the DM group were significantly lower than that in the normal control group （t=3.71,P＜0.05;t =10.09,P＜0.001）.The mCherry transfection group was strongly expressed red fluorescence,the expression of EMCN was significantly lower in retinal tissue of DM group than that in the normal control group （t=13.67,P＜0.001）.The expression of EMCN was notably upregulated in retinas of EMCN transfection group,comparing with that of DM group （t =3.18,P＜0.05）.The expression of EMCN in mCherry transfection group was similar to that in the DM group （t =2.31,P=0.08）.Initial morphologic degenerative changes were found in the DM group and mCherry transfection group,such

关 键 词：内皮黏蛋白 糖尿病视网膜病变 细胞凋亡 蛋白激酶B 半胱氨酸天冬氨酸蛋白酶3 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]