Table_5_Putative Biomarkers in Tears for Diabetic Retinopathy Diagnosis.XLSX

PurposeTear fluid biomarkers may offer a non-invasive strategy for detecting diabetic patients with increased risk of developing diabetic retinopathy (DR) or increased disease progression, thus helping both improving diagnostic accuracy and understanding the pathophysiology of the disease. Here, we assessed the tear fluid of nondiabetic individuals, diabetic patients with no DR, and diabetic patients with nonproliferative DR (NPDR) or with proliferative DR (PDR) to find putative biomarkers for the diagnosis and staging of DR.
MethodsTear fluid samples were collected using Schirmer test strips from a cohort with 12 controls and 54 Type 2 Diabetes (T2D) patients, and then analyzed using mass spectrometry (MS)-based shotgun proteomics and bead-based multiplex assay. Tear fluid-derived small extracellular vesicles (EVs) were analyzed by transmission electron microscopy, Western Blotting, and nano tracking.
ResultsProteomics analysis revealed that among the 682 reliably quantified proteins in tear fluid, 42 and 26 were differentially expressed in NPDR and PDR, respectively, comparing to the control group. Data are available via ProteomeXchange with identifier PXD033101. By multicomparison analyses, we also found significant changes in 32 proteins. Gene ontology (GO) annotations showed that most of these proteins are associated with oxidative stress and small EVs. Indeed, we also found that tear fluid is particularly enriched in small EVs. T2D patients with NPDR have higher IL-2/-5/-18, TNF, MMP-2/-3/-9 concentrations than the controls. In the PDR group, IL-5/-18 and MMP-3/-9 concentrations were significantly higher, whereas IL-13 was lower, compared to the controls.
ConclusionsOverall, the results show alterations in tear fluid proteins profile in diabetic patients with retinopathy. Promising candidate biomarkers identified need to be validated in a large sample cohort.

研究目的：泪液生物标志物可为检测糖尿病视网膜病变（Diabetic Retinopathy, DR）发病风险升高或疾病进展加速的糖尿病患者提供非侵入性策略，有助于提升诊断准确性并加深对该疾病病理生理学的认知。本研究针对非糖尿病个体、无糖尿病视网膜病变的糖尿病患者，以及合并非增殖性糖尿病视网膜病变（Nonproliferative DR, NPDR）或增殖性糖尿病视网膜病变（Proliferative DR, PDR）的糖尿病患者的泪液展开分析，以期筛选出可用于糖尿病视网膜病变诊断与分期的潜在生物标志物。
研究方法：本研究采用席默尔试纸（Schirmer test strips）采集12名对照个体与54名2型糖尿病（Type 2 Diabetes, T2D）患者的泪液样本，随后基于质谱（Mass Spectrometry, MS）的鸟枪蛋白质组学技术与基于微球的多重检测技术开展分析。同时通过透射电子显微镜、蛋白质免疫印迹（Western Blotting）及纳米追踪技术对泪液来源的小型细胞外囊泡（extracellular vesicles, EVs）进行表征分析。
研究结果：蛋白质组学分析结果显示，在泪液中可被可靠定量的682种蛋白质中，与对照组相比，非增殖性糖尿病视网膜病变组与增殖性糖尿病视网膜病变组分别有42种和26种蛋白质存在差异表达。相关数据可通过标识符为PXD033101的ProteomeXchange平台获取。通过多组比较分析，本研究还发现32种蛋白质存在显著表达变化。基因本体论（Gene Ontology, GO）注释结果显示，上述多数蛋白质与氧化应激及小型细胞外囊泡相关。本研究同时证实，泪液中富含小型细胞外囊泡。与对照组相比，合并非增殖性糖尿病视网膜病变的2型糖尿病患者泪液中白细胞介素（Interleukin, IL）-2、-5、-18，肿瘤坏死因子（Tumor Necrosis Factor, TNF）以及基质金属蛋白酶（Matrix Metalloproteinase, MMP）-2、-3、-9的浓度更高。在增殖性糖尿病视网膜病变组中，与对照组相比，IL-5、-18以及MMP-3、-9的浓度显著升高，而IL-13的浓度则显著降低。
研究结论：综上，本研究结果表明，合并视网膜病变的糖尿病患者泪液蛋白质谱存在异常。本研究筛选出的潜在候选生物标志物仍需在大样本队列中进行验证。