ASSEMBLY OF COLLAGEN FIBRILS IN CORNEAL STROMA

描述（改编自申请人摘要）：细胞外基质 在角膜基质中，是一种高度有序的结构， 维持角膜的透明度和硬度。 如何 细胞外基质的成分被组装， 细胞控制机制参与了基质的形成， 才刚刚开始被理解。 本建议的目的是研究如何 胶原蛋白的异型原纤维在人角膜中组装。 正常 人角膜细胞培养物可用作胶原蛋白的模型， 在人角膜中组装，并作为研究异型原纤维的模型系统 阵 I型缺陷的突变圆锥角膜细胞培养物， III和V胶原蛋白合成也将用于这些研究。 的 项目将使用免疫荧光显微镜，高电压 免疫电镜（HVEM）、生物化学和分子生物学 研究异型纤维形成的技术。 结构 以及I型和III型胶原蛋白的异型原纤维的形成， I型和V型胶原蛋白将使用I型特异性抗体进行研究， III和V胶原蛋白。 对纤连蛋白和II型特异性的抗体， VI胶原蛋白将用于观察这些蛋白质是否与发育中的 异型纤维 纤连蛋白和I，II， III、V和VI型胶原进入亚融合角膜基质细胞培养物的基质 将在96小时内进行随访。 免疫荧光显微术和 HVEM可用于鉴定基质原纤维的组成， 确定这些组织中胶原纤维的带型和直径， cultures. RNA和Elisa分析将被用于定量表达的量。 I型、III型和V型胶原蛋白掺入原纤维。 原位 对正常和圆锥角膜角膜钮扣的超微结构研究， 还可以将体内胶原结合模式、原纤维 异型原纤维的直径和组成， 文化 最后，研究Ⅲ型胶原在人肝细胞凋亡中的作用， 异型原纤维的形成，III型和V型的反义mRNA 胶原蛋白将被开发并插入角膜细胞中， 打开或关闭III型或V型胶原蛋白合成。

DESCRIPTION (adapted from applicant's abstract): The extracellular matrix in corneal stroma is a highly ordered structure believed to play a major role in maintaining the transparency and rigidity of the cornea. How the components of the extracellular matrix are assembled and what molecular and cellular control mechanisms are involved in the formation of the matrix are only beginning to be understood. The aim of this proposal is to study how heterotypic fibrils of collagen are assembled in human cornea. Normal human keratocyte cultures would be used as a model of collagen are assembled in human cornea and as a model system to study heterotypic fibril formation. Mutant keratoconus keratocyte cultures deficient in types I, III and V collagen synthesis would also be used in these studies. The project would use immunofluorescence microscopy, high voltage immunoelectron microscopy (HVEM), biochemical and molecular biology techniques to study the formation of heterotypic fibrils. The structure and formation of heterotypic fibrils of types I and III collagen and types I and V collagen would be studied using antibodies specific for types I, III and V collagen. Antibodies specific for fibronectin and types II and VI collagen would be used to see if these proteins interact with developing heterotypic fibrils. The incorporation of fibronectin and types I, II, III, V and VI collagen into matrix of subconfluent keratocyte cultures would be followed over a 96 hour period. Immunofluorescence microscopy and HVEM would be used to identify the composition of matrix fibrils and to determine the banding pattern and diameter of collagen fibrils in these cultures. RNA and Elisa analysis would be used to quantitate the amount of types I, III and V collagen incorporation into fibrils. In situ ultrastructural studies on normal and keratoconus corneal buttons would also be done to correlate in vivo collagen binding patterns, fibril diameter and composition of heterotypic fibrils to those observed in culture. Finally, to study the function of types III of V collagen in the formation of heterotypic fibrils, antisense mRNAs for types III and V collagen would be developed and inserted into keratocytes to selectively turn on, or off, type III or type V collagen synthesis.