REGULATION OF FIBRIL ASSOCIATED COLLAGENS IN CORNEA

成熟的、功能正常的角膜是由 几种胶原基质，如Bowman膜，基质，和 后弹力层。 透明度取决于均匀的小直径 以及基质中胶原纤维的规则间隔，并且通过以下方式获得： 使基质致密化并使胶原纤维束聚结。 这 压实伴随着原纤维相关的 胶原蛋白，XIV型。 这种胶原蛋白最近被证明有助于 体外水合胶原凝胶中原纤维的运动，因此 在体内，它可能具有类似的功能， 基质。 我们的研究表明，所有组织表达XIV型胶原蛋白 mRNA（例如，角膜上皮和基质），但不是所有的翻译mRNA (e.g.,该蛋白质不被角膜上皮表达）。 审查 角膜上皮（非表达者）如何调节转录 我们将测量蛋白质）和基质（蛋白质的表达者） 使用CAT构建体进行基因转染的转录活性 含有XIV型胶原基因启动子。 我们还将研究如何 翻译调控是通过分离和分析多核糖体来实现的 （含有可翻译mRNA的细胞内结构）和单体 （具有不可翻译的mRNA的结构）， 间质中XIV型胶原mRNA的存在。 角膜蛋白多糖的表达在发育过程中变化。 我们 我发现了XIV型胶原的4种剪接变体，它们可能与这些蛋白结合， 具有不同亲和力的蛋白聚糖。 我们将使用亲和力来测试这一点 共电泳 我们还将使用定量PCR来确定 变异在不同的发育阶段普遍存在，特别是 在获得透明度的时候。 评估功能 XIV型胶原蛋白在压实中的作用，早期角膜会受到感染， 用逆转录病毒构建体进行卵内转染， 胶原多肽 这将产生显性阴性表型， 胶原蛋白XIV分子，这应该会干扰其正常的 功能 最后，我们发现另一种纤维相关胶原蛋白，XII型， 在角膜的界面基质中是普遍存在的， 后弹力膜和后弹力膜邻接基质。 免疫组 和基因结构分析表明，在角膜中， 分子存在，一个可以提供结构稳定性，这些 矩阵 我们将使用基于PCR的策略来获得该基因的cDNA。 角膜XII型同种型，以阐明其结构。

The mature, functioning cornea results from the synthesis and assembly of several collagenous matrices, such as Bowman's membrane, the stroma, and Descemet's membrane. Transparency depends on the small uniform diameter and regular spacing of collagen fibrils in the stroma, and is acquired by compacting the stroma and coalescing the collagen fibril bundles. This compaction is accompanied by an increase in the fibril-associated collagen, type XIV. This collagen has recently been shown to facilitate the movement of fibrils in hydrated collagen gels in vitro, and therefore in vivo it may have a similar function in facilitating compaction of the stroma. Our studies suggest that all tissues express type XIV collagen mRNA (e.g., corneal epithelium and stroma), but not all translate the mRNA (e.g., the protein is not expressed by corneal epithelium). To examine how transcription is regulated by the corneal epithelium (a non-expressor of the protein), and stroma (an expressor of protein) we will measure transcriptional activity using gene transfections with CAT constructs containing the type XIV collagen gene promoter. We will also examine how translational regulation is conferred by isolating and analyzing polysomes (intracellular structures containing translatable mRNAs) and monosomes (structures with non-translatable mRNAs) from corneal epithelia and stromas for the presence of type XIV collagen mRNAs. The expression of corneal proteoglycans varies during development. We have found 4 splice variants of type XIV collagen that may bind these proteoglycans with different affinities. We will test this using affinity coelectrophoresis. We will also use quantitative PCR to determine which variants are prevalent at different developmental stages, especially around the time that transparency is acquired. To assess the functional role of type XIV collagen in compaction, early corneas will be infected in ovo with a retroviral construct that synthesizes a truncated type XIV collagen polypeptide. This will create a dominant negative phenotype for the collagen XIV molecule, which should interfere with its normal function. Lastly, we have found that another fibril-associated collagen, type XII, is prevalent in the interfacial matrices of the cornea, where Bowman's membrane and Descemet's membrane adjoin the stroma. Immunohistochemistry and gene structural analysis suggest that in cornea a new isoform of the molecule exists, one which may provide structural stability to these matrices. We will use a PCR based strategy to obtain a cDNA for the corneal type XII isoform, to elucidate its structure.