角膜上皮基底膜的组装是由上皮细胞在伤口愈合过程中与角膜成纤维细胞协调完成的。
Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing.
机构信息
Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio.
出版信息
Mol Vis. 2023 May 20;29:68-86. eCollection 2023.
PURPOSE
To understand which cell types, either alone or in combination, contribute to the assembly of the epithelial basement membrane (BM) during corneal wound healing.
METHODS
A 3D corneal organotypic model and an in situ rabbit photorefractive keratectomy (PRK) model were used in this study. The 3D corneal organotypic model was established by culturing the rabbit corneal epithelial cells with either corneal fibroblasts or myofibroblasts embedded in collagen type I for 18 days. Corneal fibroblasts were isolated from fresh rabbit corneas, and the myofibroblasts were derived either directly from bone marrow or differentiated from corneal fibroblasts. Immunocytochemistry for alpha-smooth muscle actin (SMA), vimentin, desmin, and vinculin markers confirmed well-differentiated myofibroblasts. Immunohistochemistry was performed in cryofixed sections for BM markers, including laminin alpha-5, laminin beta-3, perlecan, nidogen-1, and collagen type IV. Specimens were also examined with transmission electron microscopy (TEM). Corneas were collected from rabbits after -3 diopter (D) PRK at different time points after surgery, with four corneas at each time point in each group. Cryofixed corneal sections were stained for vimentin, alpha-SMA, and nidogen-1.
RESULTS
The formation of an epithelial BM with expression of laminin alpha-5, laminin beta-3, perlecan, nidogen-1, and collagen IV was observed at the interface between the corneal epithelial cells and corneal fibroblasts. TEM images further confirmed the presence of epithelial BM in organotypic cultures of epithelial cells and corneal fibroblasts. No epithelial BM was observed in cultures of corneal epithelial cells and myofibroblasts (cornea or bone marrow derived), corneal epithelial cells alone, or corneal fibroblasts alone. In rabbit corneas after -3D PRK, a strong association was observed between the regenerating epithelial BM and the presence of corneal fibroblasts at the site of epithelial BM generation.
CONCLUSIONS
The corneal epithelial BM assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing.
目的
了解在角膜伤口愈合过程中，是单独的哪种细胞类型，还是多种细胞类型的组合，促成了上皮基底膜（BM）的组装。
方法
本研究使用了三维角膜器官型模型和原位兔光折射性角膜切削术（PRK）模型。三维角膜器官型模型是通过将兔角膜上皮细胞与嵌入 I 型胶原的角膜成纤维细胞或肌成纤维细胞共培养 18 天而建立的。角膜成纤维细胞从新鲜兔角膜中分离得到，肌成纤维细胞则直接来源于骨髓或由角膜成纤维细胞分化而来。免疫细胞化学染色用于检测α-平滑肌肌动蛋白（SMA）、波形蛋白、结蛋白和纽蛋白的标志物，以确认分化良好的肌成纤维细胞。冷冻固定切片进行 BM 标志物免疫组织化学染色，包括层粘连蛋白α-5、层粘连蛋白β-3、蛋白聚糖、巢蛋白-1 和 IV 型胶原。标本也通过透射电子显微镜（TEM）进行了检查。在手术后不同时间点，对接受 -3 屈光度（D）PRK 的兔眼收集角膜，每组每个时间点有 4 只兔眼。冷冻固定角膜切片用波形蛋白、α-SMA 和巢蛋白-1 染色。
结果
在角膜上皮细胞和角膜成纤维细胞之间的界面处观察到形成具有层粘连蛋白α-5、层粘连蛋白β-3、蛋白聚糖、巢蛋白-1 和 IV 型胶原表达的上皮 BM。TEM 图像进一步证实了上皮细胞和角膜成纤维细胞的器官型培养中存在上皮 BM。在角膜上皮细胞和肌成纤维细胞（源自角膜或骨髓）、单独的角膜上皮细胞或单独的角膜成纤维细胞的培养物中未观察到上皮 BM。在接受 -3D PRK 的兔角膜中，在再生的上皮 BM 与上皮 BM 生成部位的角膜成纤维细胞之间观察到强烈的关联。
结论
在伤口愈合过程中，角膜上皮 BM 的组装是由上皮细胞与角膜成纤维细胞协调介导的。
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