Abstract：
Prolonged hyperglycemia during diabetes mellitus (DM) is associated with severe complications that may affect both the anterior and posterior ocular segments, leading to impaired vision or blindness. The cornea is a vital part of the eye that has a dual role as a protective transparent barrier and as a major refractive structure and is likewise negatively affected by hyperglycemia in DM. Understanding the cellular and molecular mechanisms underlying the phenotypic changes associated with DM is critical to developing targeted therapies to promote tissue integrity. In this proof-of-concept study, we applied a cell sheet-based approach to generate stacked constructs of physiological corneal thickness using primary human corneal fibroblasts isolated from cadaveric control (healthy), Type 1 DM and Type 2 DM corneal tissues. Self-assembled corneal stromal sheets were generated after 2 weeks in culture, isolated, and subsequently assembled to create stacked constructs, which were evaluated using transmission electron microscopy. Analysis of gene expression patterns revealed significant downregulation of fibrotic markers, α-smooth muscle actin, and collagen type 3, with stacking in Type 2 DM constructs when compared to controls. IGF1 expression was significantly upregulated in Type 2 DM constructs compared to controls with a significant reduction induced by stacking. This study describes the development of a thicker, self-assembled corneal stromal construct as a platform to evaluate phenotypic differences associated with DM-derived corneal fibroblasts and enable the development of targeted therapeutics to promote corneal integrity.
摘要：
糖尿病（DM）期间的长期高血糖与可能影响前眼和后眼节的严重并发症有关。导致视力受损或失明。角膜是眼睛的重要部分,其具有作为保护性透明屏障和作为主要屈光结构的双重作用,并且同样受到DM中的高血糖的负面影响。了解与DM相关的表型变化的细胞和分子机制对于开发靶向治疗以促进组织完整性至关重要。在这个概念验证研究中,我们应用基于细胞片的方法，使用从尸体对照（健康）分离的原代人角膜成纤维细胞产生生理角膜厚度的堆叠构建体，1型DM和2型DM角膜组织。培养2周后生成自组装角膜基质片，孤立的,随后组装以创建堆叠结构，使用透射电子显微镜进行评估。基因表达模式分析显示纤维化标志物显著下调，α-平滑肌肌动蛋白，和3型胶原蛋白，与对照相比，在2型DM构建体中堆叠。与对照相比,IGF1表达在2型DM构建体中显著上调,通过堆叠诱导显著降低。这项研究描述了一种较厚的发展，自组装角膜基质构建体作为评估与DM衍生角膜成纤维细胞相关的表型差异的平台，并能够开发靶向治疗以促进角膜完整性。
