Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine, Shanghai, 200090, China; Department of Ophthalmology, Tongji Hospital, School of Medicine, Tongji University, Shanghai, 200065, China.
Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine, Shanghai, 200090, China.
Exp Eye Res. 2021 Nov;212:108794. doi: 10.1016/j.exer.2021.108794. Epub 2021 Oct 14.
Diabetic Keratopathy (DK) is one of the significant complications of type II diabetes (T2DM) with pathogenesis not yet clarified. Since hyperglycemia is able to change the protein components contained in plasma exosomes, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as feasible to analyze the expression of plasma exosomal proteins in patients with T2DM and non-diabetic patients respectively, find critical biological markers, and explore the mechanism of DK as well as potential therapeutic targets.
Blood and clinical information of corneal epithelial injury in a diabetic group (the study group) and a non-diabetic group (the control group), who were patients admitted to the Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine from July 2020 to November 2020, were collected. The qEV size exclusion method was adopted to separate exosomes from plasma. The exosomes were then identified through transmission electron microscopy (TEM), nanoparticle tracking analyzer (NTA), and Western blot. The plasma exosomes of the study group and the control group were quantitatively analyzed by proteomics. A bioinformatics method is utilized to screen differential proteins and the expression of the differential proteins was verified by Western blot.
TEM indicated that the exosomes had a double-concave disc-like appearance, with a size of about 100 nm, and Western blot expressed as CD63 and TSG101. The plasma exosomes of the study group and the control group were analyzed by quantitative proteomics with a total number of 952 proteins detected of which 245 proteins existed in the ExoCarta exosomal protein database. Through adoption of P-value to screen credible differential proteins, the heat map displayed 28 differential proteins, 7 upregulated proteins, and 21 downregulated proteins; the volcano map displayed 7 upregulated proteins and 22 downregulated proteins; the PPI interaction map displayed 12 upregulated proteins and 18 downregulated proteins. Through GO enrichment analysis, it was identified that the differential protein participated in the main biological processes and was involved in regulating the cell's stimulation response to insulin, the insulin receptor signaling pathway, and the activity of glycosylphosphatidylinositol phospholipase D as well as anti-oxidation. The enriched cell components include main components such as exosomes, blood particles, and cytoplasm. KEGG enrichment analysis indicated that the target protein FLOT2 was mainly concentrated in insulin-related signaling pathways. Western blot indicated that the expression of FLOT2 in the study group was lower compared with the control group while the expression of Exo70 was higher.
Proteomic analysis of the study group and the control group displayed a variety of proteins in plasma exosomes. The downregulated protein FLOT2 in the study group was closely related to the occurrence, development, and complication of DK in T2DM patients. The expression status of plasma FLOT2 protein in T2DM patients is expected to be a biomarker for diagnosing and monitoring of DK.
糖尿病性角膜病变（DK）是 2 型糖尿病（T2DM）的一种严重并发症，其发病机制尚不清楚。由于高血糖能够改变血浆外泌体中所含的蛋白质成分，因此液相色谱-串联质谱（LC-MS/MS）被认为是可行的方法，可以分别分析 T2DM 患者和非糖尿病患者的血浆外泌体蛋白的表达，找到关键的生物标志物，并探索 DK 的发病机制以及潜在的治疗靶点。
收集 2020 年 7 月至 2020 年 11 月同济大学附属杨浦医院眼科收治的糖尿病组（研究组）和非糖尿病组（对照组）角膜上皮损伤患者的血液和临床信息。采用 qEV 排阻大小法从血浆中分离外泌体。通过透射电子显微镜（TEM）、纳米颗粒跟踪分析仪（NTA）和 Western blot 对外泌体进行鉴定。采用蛋白质组学方法对研究组和对照组的血浆外泌体进行定量分析。采用生物信息学方法筛选差异蛋白，并通过 Western blot 验证差异蛋白的表达。
TEM 显示外泌体呈双凹盘状，大小约 100nm，Western blot 表达 CD63 和 TSG101。研究组和对照组的血浆外泌体通过定量蛋白质组学分析，共检测到 952 种蛋白质，其中 245 种蛋白质存在于 ExoCarta 外泌体蛋白质数据库中。通过 P 值筛选可信的差异蛋白，热图显示 28 种差异蛋白，7 种上调蛋白，21 种下调蛋白；火山图显示 7 种上调蛋白和 22 种下调蛋白；PPI 相互作用图显示 12 种上调蛋白和 18 种下调蛋白。通过 GO 富集分析，发现差异蛋白主要参与细胞的刺激反应、胰岛素受体信号通路、糖基磷脂酰肌醇磷脂酶 D 的活性和抗氧化等生物学过程。细胞富集的主要成分包括外泌体、血液颗粒和细胞质等主要成分。KEGG 富集分析表明，FLOT2 靶蛋白主要集中在胰岛素相关信号通路中。Western blot 显示研究组 FLOT2 蛋白表达低于对照组，而 Exo70 蛋白表达高于对照组。
研究组和对照组的血浆外泌体蛋白质组学分析显示，血浆外泌体中存在多种蛋白质。研究组下调蛋白 FLOT2 与 T2DM 患者 DK 的发生、发展和并发症密切相关。T2DM 患者血浆 FLOT2 蛋白的表达状态有望成为诊断和监测 DK 的生物标志物。
