REGULATION OF FIBRIL ASSOCIATED COLLAGENS IN CORNEA

成熟的、有功能的角膜是由合成和组装 几种胶原基质，如鲍曼膜、基质和 Descemet膜。透明度取决于细小的均匀直径 和间质中规则间隔的胶原纤维，并通过 使基质紧凑，并使胶原纤维束结合。这 压实伴随着纤维相关纤维的增加。 胶原蛋白，XIV型。这种胶原蛋白最近被证明可以促进 纤维在水合胶原凝胶中的运动，因此 在体内，它可能具有类似的功能，促进血管紧实 斯特尔玛。我们的研究表明，所有组织都表达XIV型胶原 M RNA(例如，角膜上皮和基质)，但不是全部翻译该m RNA (例如，该蛋白不在角膜上皮细胞中表达)。检视 转录如何受角膜上皮(一种非表达因子)的调节 和基质(一种蛋白质的表达因子)，我们将测量 利用CAT构建的基因转染法实现转录活性 含有XIV型胶原基因启动子。我们还将研究如何 翻译调节是通过分离和分析多聚体来实现的 (含有可翻译的mRNA的细胞内结构)和单体 (含有不可翻译的mRNA的结构)来自角膜上皮和 基质中存在XIV型胶原mRNAs。 角膜蛋白多糖的表达在发育过程中会发生变化。我们 我发现了XIV型胶原的4种剪接变体，它们可能结合这些 具有不同亲和力的蛋白多糖。我们将使用亲和力测试这一点 共电泳法。我们还将使用定量聚合酶链式反应来确定 变异体在不同的发育阶段很普遍，尤其是 大约在获得透明度的时候。要评估功能 XIV型胶原在致密中的作用，早期角膜会感染 用逆转录病毒构建的卵子合成截短型XIV 胶原蛋白多肽。这将创造一个显性的负面表型 XIV胶原蛋白分子，这应该会干扰其正常 功能。 最后，我们发现另一种纤维相关的胶原蛋白，XII型， 普遍存在于角膜的界面基质中，鲍曼的 膜和Descemet膜毗邻基质。免疫组织化学 基因结构分析表明，在角膜中，一种新的亚型 分子的存在，可以为这些分子提供结构稳定性 矩阵。我们将使用基于聚合酶链式反应的策略来获得 角膜XII亚型，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.