TYPE XVIII COLLAGEN/ENDOSTATIN FUNCTION IN C ELEGANS

本研究的目的是确定XVIII型胶原及其内皮抑素结构域的正常功能，并阐明其作用的分子机制。内皮抑素具有很强的抗血管生成肿瘤治疗的潜力，但人们对其或其衍生的XVIII型胶原的正常功能知之甚少。已在线虫中鉴定出XVIII型胶原同源物Cle1，并将利用该模型系统的优势来分析其在发育中的功能，并确定它可能与之相互作用的其他分子。细胞周期蛋白-1的突变可能导致细胞和轴突引导的缺陷，并可能阻止某些细胞的凋亡性死亡。异位表达的内皮抑素结构域导致特定细胞的导向缺陷和凋亡性死亡。哺乳动物和线虫的内皮抑素结构域是否在功能上相同，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生血管的影响来测试。人类内皮抑素序列将在线虫中表达，以确定它们是否可以挽救cle-1突变并导致预期的异位表型。将使用GFP标记和谱系分析来确定受cle-1突变和异位表达影响的特定细胞的身份。通过控制异位内皮抑素表达的时间和位置，我们将检查它对特定细胞的影响要求。将产生抗体来确定三种CLE-1亚型的定位，并评估它们在突变体中是如何受到影响的。这些抗体还将用于分析从CLE-1释放的内皮抑素结构域和蛋白质的其他特征。将产生更多的CLE-1突变并对其进行鉴定。将产生增强或抑制cle-1突变或异位表达表型的突变，并确定它们所影响的基因。这些基因可能编码与XVIII型胶原和/或内皮抑素相互作用的分子，例如其他基质蛋白或受体，或者是它们正常功能所必需的，例如信号通路。对这些基因的进一步研究可能确定XVIII型胶原和内皮抑素发挥作用的机制。这些研究的结果可能会提示内皮抑素治疗可能的副作用，应该仔细监测，以及可能的方法来增强其有效性。

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.