糖尿病视网膜病变患者循环外泌体中炎症相关蛋白S100A8的表达  

Circulating exosomal inflammation-related protein S100A8 as a potential biomarker for the severity of diabetic retinopathy

作　　者：于荣国 张慧 张晓敏 邵先锋 李筱荣 Yu Rongguo;Zhang Hui;Zhang Xiaomin;Shao Xianfeng;Li Xiaorong(Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin International Joint Research and Development Centre of Ophthalmology and Vision Science,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院、眼视光学院、眼科研究所,天津市视网膜功能与疾病重点实验室,天津市眼科学与视觉科学国际联合研究中心,300384

出　　处：《中华眼底病杂志》2021年第1期32-39,共8页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金(81870675、81800825);天津市教委科研计划项目(2019ZD030);天津市科技支撑重点项目(20YFZCSY00990);天津市视网膜功能与疾病重点实验室自主与开放课题(2019tjswmm001)。

摘　　要：目的观察糖尿病视网膜病变(DR)患者血浆外泌体、微囊泡(MV)、血浆和玻璃体中炎症相关蛋白S100A8的表达,并于糖尿病大鼠模型中进行验证,初步探讨其在DR发生和发展中的作用。方法病例对照研究和基础研究。2018年9月至2019年12月于天津医科大学眼科医院就诊的2型糖尿病患者、住院行玻璃体切割手术患者以及同期健康体检者共计73名纳入研究。其中,采集血浆32名,收集玻璃体液41名,并据此分为血浆样本研究队列和玻璃体样本研究队列。将受试者分为未发生眼底改变的单纯糖尿病组(DM组)、非增生型DR组(NPDR组)和增生型DR组(PDR组);健康体检者作为正常对照组(NC组)。玻璃体样本研究队列中对照组为黄斑前膜或黄斑裂孔患者玻璃体液。超速离心法分离血浆外泌体和MV。采用透射电子显微镜、纳米粒度分析仪、蛋白质免疫印迹法(Western blot)鉴定外泌体和MV。采用酶联免疫吸附试验法测定S100A8质量浓度。18只健康雄性Brown Norway大鼠经随机数字表法分为正常对照组和糖尿病组,每只9只。糖尿病组大鼠经链脲佐菌素诱导建立糖尿病模型。建模后5个月采用免疫组织化学染色、Western blot检测正常对照组、糖尿病组大鼠视网膜S100A8的表达情况。两组间计量数据比较采用t检验;多组计量数据比较采用单因素方差分析。结果血浆中成功分离得到具有各自特征的外泌体和MV。PDR组患者血浆外泌体和玻璃体S100A8浓度均高于NPDR组、DM组、NC组,差异有统计学意义(P=0.039、0.020、0.002、0.002,P<0.000、<0.000 )。血浆样本队列研究4个组受试者血浆、血浆MV的S100A8质量浓度总体比较,差异无统计学意义(F=0.283、0.015 ,P=0.836、0.996 )。免疫组织化学染色结果显示,糖尿病组大鼠视网膜神经节细胞、双极细胞、视锥视杆细胞和血管内皮细胞均表达S100A8蛋白。与正常对照组大鼠比较,糖尿病组大鼠视Objective To observe the expression of S100A8 in plasma exosomes,microvesicles(MV),plasma and vitreous in patients with diabetic retinopathy(DR),and verify it in a diabetic rat model,and to preliminarily explore its role in the occurrence and development of DR.Methods A case-control study.From September 2018 to December 2019,a total of 73 patients with type 2 diabetes,hospitalized patients undergoing vitrectomy,and healthy physical examinations in the Tianjin Medical University Eye Hospital were included in the study.Among them,plasma were collected from 32 patients and vitreous fluid were collected from 41 patients,which were divided into plasma sample research cohort and vitreous sample research cohort.The subjects were divided into simple diabetes group(DM group),non-proliferative DR group(NPDR group)and proliferative DR group(PDR group)without fundus changes;healthy subjects were regarded as normal control group(NC group).In the study cohort of vitreous samples,the control group was the vitreous humor of patients with epimacular membrane or macular hole.Plasma exosomes and micro vesicles(MVs)were separated using ultracentrifiigation.Transmission electron microscopy,nanometer particle size analyzer and Western blot(WB)were used to characterize exosomes and MVs.The mass concentration of SI00A8 was determined by enzyme-linked immunosorbent assay.Eighteen healthy male Brown Norway rats were divided into normal control group and diabetic group with 9 rats in each group by random number table method.The rats of diabetes group were induced by streptozotocin to establish diabetic model.Five months after modeling,immunohistochemical staining and WB were used to detect the expression of S100A8 in the retina of rats in the normal control group and the diabetes group,t test was used for the comparison of measurement data between the two groups.Singlefactor analysis of variance were used for the comparison of multiple groups of measurement data.parison of measurement data between the two groups.Single-factor analysis of v

关 键 词：糖尿病视网膜病变 外泌体 玻璃体 钙粒蛋白A 炎症 

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