ASSEMBLY OF COLLAGEN FIBRILS IN CORNEAL STROMA

描述(改编自申请人的摘要)：细胞外基质 在角膜基质中，一种高度有序的结构被认为是发挥主要作用的 在维持角膜的透明度和刚性方面起到了重要作用。怎么了？ 细胞外基质的成分是组装的，什么分子和 细胞控制机制参与了基质的形成 只是刚刚开始被理解。这项建议的目的是研究如何 异型胶原纤维在人的角膜中组装。正常 人角膜基质细胞培养将被用作胶原蛋白的模型 在人角膜中组装并作为研究异型纤维的模型系统 队形。缺乏I型的突变圆锥角膜细胞培养物， III型和V型胶原的合成也将用于这些研究。这个 该项目将使用免疫荧光显微镜，高压 免疫电子显微镜、生物化学和分子生物学 研究异型纤维形成的技术。该结构 形成I型和III型胶原的异型纤维和 将使用针对I型的抗体来研究I型和V型胶原， III型和V型胶原。纤维连接蛋白和II型和II型特异性抗体 VI胶原蛋白将被用于观察这些蛋白质是否与发育中的 异型纤维。纤维连接蛋白与I型、II型、 III、V、VI型胶原在亚融合培养的角膜基质中的应用 将在96小时内被跟踪。免疫荧光显微镜和 高能电子显微镜将用于鉴定基质纤维的组成，并 确定这些组织中胶原纤维的条带模式和直径 文化。RNA和ELISA法将被用来定量 I型、III型和V型胶原掺入纤维中。就地 对正常和圆锥角膜纽扣的超微结构研究将 也可以用来关联体内的胶原结合模式、纤维 异型纤维的直径和组成与在 文化。最后，研究V型胶原蛋白III型在血管内皮细胞中的作用。 III型和V型异型纤维、反义mRNAs的形成 胶原蛋白将被开发并插入角质形成细胞中，以选择性地 打开或关闭III型或V型胶原合成。

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.