TYPE XVIII COLLAGEN/ENDOSTATIN FUNCTION IN C ELEGANS

本研究的目的是确定 XVIII 型胶原蛋白及其内皮抑素结构域的正常功能，并阐明它们作用的分子机制。 内皮抑素具有作为抗血管生成肿瘤疗法的巨大潜力，但对其或其来源的 XVIII 型胶原蛋白的正常功能知之甚少。 XVIII 型胶原蛋白同源物 cle-1 已在线虫中鉴定出来。该模型系统的优势将用于分析其发育过程中的功能，并鉴定可能与其相互作用的其他分子。 cle-1 突变可导致细胞和轴突引导缺陷，并可能阻止某些细胞的凋亡。 cle-1 内皮抑素结构域的异位表达会导致特定细胞的引导缺陷和凋亡。 将通过比较重组蛋白对人内皮细胞迁移和凋亡以及对大鼠角膜新血管形成的影响来测试哺乳动物和线虫内皮抑素结构域在功能上是否等效。 人内皮抑素序列将在秀丽隐杆线虫中表达，以确定它们是否可以挽救 cle-1 突变并引起预期的异位表型。 受 cle-1 突变和异位表达影响的特定细胞的身份将使用 GFP 标记和谱系分析来确定。 通过控制异位内皮抑素表达的时间和位置，将检查其影响特定细胞的要求。 将生成抗体来确定三种 CLE-1 同工型的定位并评估它们在突变体中受到的影响。 这些抗体还将用于分析 CLE-1 内皮抑素结构域的释放以及该蛋白质的其他特征。 将生成并表征其他 cle-1 突变。 将产生增强或抑制 cle-1 突变体或异位表达表型的突变，并识别它们影响的基因。 这些基因可能编码与 XVIII 型胶原蛋白和/或内皮抑素相互作用的分子，例如其他基质蛋白或受体，或者是其正常功能所必需的，例如信号通路。对这些基因的进一步研究可能会确定 XVIII 型胶原蛋白和内皮抑素发挥作用的机制。这些研究的结果可能表明内皮抑素治疗可能存在副作用，应仔细监测这些副作用，并提出增强其有效性的潜在方法。

The goals of this research are to determine the normal functions of type XVIII collagen and its endostatin domain, and to elucidate the molecular mechanisms by which they act. Endostatin has strong potential for use as an anti-angiogenic tumor therapy, however very little is known about the normal functions of it, or type XVIII collagen, from which it is derived. A type XVIII collagen homologue, cle-1, has been identified in the nematode C. elegans and the advantages of this model system will be exploited to analyze its function in development and to identify other molecules with which it may interact. Mutations in cle-1 can cause defects in cell and axon guidance, and may block apoptotic death of certain cells. Ectopic expression of the endostatin domain of cle-1 causes guidance defects and apoptotic death of particular cells. Whether mammalian and C. elegans endostatin domains are functionally equivalent will be tested by comparing the effects of recombinant proteins on migration and apoptosis of human endothelial cells, and on neovascularization of rat corneas. Human endostatin sequences will be expressed in C. elegans to determine if they can rescue cle-1 mutations and cause the expected ectopic phenotypes. The identities of specific cells affected by cle-1 mutations and ectopic expression will be determined using GFP markers and lineage analyses. By controlling the timing and location of ectopic endostatin expression, requirements for it to affect particular cells will be examined. Antibodies will be generated to determine the localization of the three CLE-1 isoforms and to assess how they are affected in mutants. The antibodies will also be used to analyze release of the endostatin domain from CLE-1 and other characteristics of the protein. Additional cle-1 mutations will be generated and characterized. Mutations that enhance or suppress cle-1 mutant or ectopic expression phenotypes will be generated and the genes that they affect identified. These genes may encode molecules that interact with type XVIII collagen and/or endostatin, e.g. other matrix proteins or receptors, or are necessary for their normal function, e.g. signaling pathway. Further studies of these genes may identify the mechanisms by which type XVIII collagen and endostatin exert their effects. The results of these studies may suggest possible side effects of endostatin treatment, that should be carefully monitored, and potential means to augment its effectiveness.