Ruggiero F, Burillon C, Garrone R
Institut de Biologie et Chimie des Protéines, Université Lyon, France.
Invest Ophthalmol Vis Sci. 1996 Aug;37(9):1749-60.
The stroma of the developing cornea is a highly organized extracellular matrix formed essentially by uniform, small-diameter collagen fibrils with constant interfibrillar spacing. Unlike the fibrillogenesis of chicken cornea, the assembly and maturation of human corneal fibrils have been poorly investigated. In the current study, the authors aimed to ascertain the heterotypic organization (collagens I and V) of the human corneal fibrils at the supramolecular level. To gain more insight into the molecular structure of collagen V, its cellular source, and its role in fibrillogenesis, the authors used cultured human corneal fibroblasts.
The structure of human corneal stroma after brief homogenization of the tissue was analyzed by immunogold labeling using specific polyclonal antibodies and rotary shadowing. Biochemical, electron microscopic, and immunolabeling approaches were used to investigate the collagen fibril formation and the extracellular matrix synthesis using human corneal fibroblasts grown in culture as a model system.
The authors showed that in human corneal stroma, collagen I is distributed uniformly along the striated fibrils, in contrast to collagen V, which could be identified only at sites at which the fibrils partially were disrupted. Rotary shadowing observations of the homogenate revealed that collagen VI, a major component of the human cornea, was associated closely with the collagen fibril surface. Corneal fibroblasts synthesize and deposit a collagenous matrix with fibrils resembling those of the human cornea in appearance and collagen composition. Biochemical data indicate that a high concentration (20% to 30%) of collagen V is synthesized by stromal fibroblasts and that collagen V molecules are processed similarly to matrix forms in which the extension peptides are retained on the molecules.
The heterotypic nature (collagens I and V) of human corneal fibrils was determined. Results indicate that human corneal fibroblasts synthesize the major collagen types in human cornea (collagens I, V, and VI) and express all the posttranslational equipment for correct collagen molecular assembly and processing in a manner that closely resembles the situation in situ, offering the opportunity for more detailed study of this process, which is essential for optical transparency.
发育中的角膜基质是一种高度有序的细胞外基质，主要由直径均匀、较小的胶原纤维组成，纤维间间距恒定。与鸡角膜的纤维形成不同，人类角膜纤维的组装和成熟过程研究较少。在本研究中，作者旨在确定人类角膜纤维在超分子水平上的异型组织（I型和V型胶原）。为了更深入了解V型胶原的分子结构、细胞来源及其在纤维形成中的作用，作者使用了培养的人类角膜成纤维细胞。
通过使用特异性多克隆抗体的免疫金标记和旋转阴影技术，对组织短暂匀浆后的人类角膜基质结构进行分析。采用生化、电子显微镜和免疫标记方法，以培养的人类角膜成纤维细胞作为模型系统，研究胶原纤维的形成和细胞外基质的合成。
作者发现，在人类角膜基质中，I型胶原沿横纹纤维均匀分布，而V型胶原仅在纤维部分断裂的部位才能被识别。匀浆的旋转阴影观察显示，人类角膜的主要成分VI型胶原与胶原纤维表面紧密相关。角膜成纤维细胞合成并沉积一种胶原基质，其纤维在外观和胶原组成上与人类角膜的纤维相似。生化数据表明，基质成纤维细胞合成高浓度（20%至30%）的V型胶原，且V型胶原分子的加工方式与保留延伸肽的基质形式相似。
确定了人类角膜纤维的异型性质（I型和V型胶原）。结果表明，人类角膜成纤维细胞合成人类角膜中的主要胶原类型（I型、V型和VI型胶原），并表达所有用于正确组装和加工胶原分子的翻译后机制，其方式与原位情况非常相似，为更详细研究这一对于光学透明性至关重要的过程提供了机会。
