Department of Ophthalmology and Visual Science, Federal University of São Paulo, São Paulo, Brazil, UNIFESP.
Nephrology Division, Federal University of São Paulo, São Paulo, Brazil, UNIFESP.
Mol Vis. 2024 Sep 2;30:279-288. eCollection 2024.
This study aimed to characterize the inflammatory mediators present in the tear film of patients with keratoconus (KC). It also aimed to investigate the gene expression of these mediators in corneal epithelial cells and their immune activity in conjunctival epithelial cells in patients with KC compared to a control group.
This transversal study included 30 patients with KC and 23 control group participants. Tear samples were collected by washing the ocular surface with 60 μL of sterile buffered saline solution. The levels of interleukin IL-5, IL-13, IL-2, IL-6, IL-10, interferon-gamma, tumor necrosis factor-alpha, and IL-4 were measured using a LEGEND plex HU Th1/Th2 panel kit and analyzed using flow cytometry. Corneal epithelial samples were obtained via manual keratectomy from KC patients scheduled for corneal crosslinking and from individuals scheduled for photorefractive keratectomy (control group). These samples were immediately stored at -70 °C for mRNA extraction and subsequent reverse transcription polymerase chain reaction analysis to measure and gene expression. Conjunctival epithelium samples were collected using impression cytology and analyzed using immunohistochemistry and confocal microscopy to detect IL-5 and IL-6 immunoreactions.
Our study found no statistically significant differences in the tear film cytokine concentrations between the two groups. In addition, the gene expression of and in the corneal epithelium was higher in the KC group than in the control group, with showing a 50% increase and IL-6 showing a 20% increase. Immunohistochemical analysis revealed a greater immunostaining of IL-5 and IL-6 in the conjunctival epithelium of patients with KC compared to the control group.
In this study, despite higher levels of IL-5 and IL-6 in the tear film of patients with KC, there was no statistically significant difference compared to the control group. However, there was heightened immune activity in the corneal and conjunctival epithelial cells of patients with KC based on IL-5 and IL-6 gene expression and their immunodetection, respectively.
本研究旨在描述圆锥角膜（KC）患者泪膜中存在的炎症介质，并探讨与对照组相比，KC 患者角膜上皮细胞中这些介质的基因表达及其在结膜上皮细胞中的免疫活性。
本横断面研究纳入 30 名 KC 患者和 23 名对照组参与者。通过用 60μL 无菌缓冲盐水溶液冲洗眼表收集泪液样本。使用 LEGENDplex HU Th1/Th2 面板试剂盒测量白细胞介素（IL）-5、IL-13、IL-2、IL-6、IL-10、干扰素-γ、肿瘤坏死因子-α和 IL-4 的水平，并通过流式细胞术进行分析。从计划行角膜交联术的 KC 患者和计划行准分子激光角膜切削术（对照组）的个体中通过手动角膜切除术获得角膜上皮样本。这些样本立即储存在-70°C，用于提取 mRNA 并随后进行逆转录聚合酶链反应分析，以测量 和 基因表达。使用印迹细胞学收集结膜上皮样本，并通过免疫组化和共聚焦显微镜分析检测 IL-5 和 IL-6 免疫反应。
我们的研究未发现两组间泪膜细胞因子浓度存在统计学差异。此外，KC 组角膜上皮中 和 的基因表达高于对照组，其中 增加了 50%，IL-6 增加了 20%。免疫组织化学分析显示，与对照组相比，KC 患者的结膜上皮中 IL-5 和 IL-6 的免疫染色更强。
在这项研究中，尽管 KC 患者的泪膜中 IL-5 和 IL-6 水平较高，但与对照组相比，并无统计学差异。然而，基于 IL-5 和 IL-6 的基因表达及其免疫检测，KC 患者的角膜和结膜上皮细胞的免疫活性增加。
