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Abstract：
Currently, in vitro cultured corneal epithelial transplantation is effective in treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. In this study, the traditional amniotic membrane (AM) was modified, and mesenchymal stem cells (MSCs) were inoculated into the ultra-thin amniotic membrane (UAM) stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier, that could effectively simulate the limbal stem cells (LSCs) microenvironment. The structure of different carriers cultured tissue-engineered corneal epithelium and the managed rabbit LSCD model corneas were observed through hematoxylin-eosin staining. Cell phenotypes were evaluated through fluorescence staining, Western blotting, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. In conclusion, the UAM-MSCs vector we constructed could better simulate the limbal microenvironment; the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype.
摘要：
目前,体外培养的角膜上皮移植可有效治疗角膜缘干细胞功能障碍（LSCD）。选择载体对于通过组织工程构建角膜上皮至关重要。在这项研究中,传统的羊膜（AM）被修改，并将间充质干细胞(MSCs)接种到超薄羊膜(UAM)基质中,构建新型UAM-MSC组织工程角膜上皮载体,能有效模拟角膜缘干细胞(LSCs)微环境。通过苏木精-伊红染色观察不同载体培养的组织工程角膜上皮和管理的兔LSCD模型角膜的结构。通过荧光染色评估细胞表型，西方印迹,和RT-qPCR。此外,使用RT-qPCR评估与抗新生血管形成相关的细胞连接基因和表达标记。通过电子显微镜观察角膜上皮细胞连接。通过质谱分析组织工程化角膜上皮培养基。通过LSCs在UAM-MSCs上扩增的组织工程角膜上皮细胞具有良好的透明度。同时,祖细胞(K14，PNCA，p63）和使用UAM-MSC构建的组织工程化角膜上皮中的角膜上皮（PAX6）基因表达高于UAM扩增的角膜上皮细胞和去上皮化羊膜。电子显微镜显示移植有UAM-MSCs的角膜上皮细胞紧密相连。总之,构建的UAM-MSCs载体能较好地模拟角膜缘的微环境,抗新血管形成特性，更紧密的细胞间连接，与自然角膜上皮组织表型更相似。
