Maspin in the Cornea

角膜内的 Maspin

基本信息

批准号：
6508796
负责人：
Sally S. Twining
金额：
$ 28.78万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-08-01 至 2006-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): bMaspin is a member of the serine proteinase inhibitor superfamily that has been studied primarily for its tumor suppressor function in carcinoma cells. Like many epithelial cells, normal corneal epithelial and endothelial cells synthesize maspin. Surprisingly, normal human corneal stromal cells also synthesize maspin, the only non-epithelial cell type known to synthesize this molecule. In culture, as passaged corneal stromal cells develop properties resembling wound fibroblasts, they lose their ability to synthesize maspin, reminiscent of the loss by metastatic carcinoma cells. However, corneal stromal fibroblasts respond to treatment with exogenous maspin by increased adhesion to types I and IV collagens, fibronectin and laminin. Studying maspin's function in the cornea is particularly important since its ability to regulate matrix attachment and cell motility may play a critical role in such events as regulation of stromal wound healing and prevention of tumorigenesis and angiogenesis. This proposal specifically addresses the hypothesis that the increase in adhesion of corneal stromal fibroblasts to type I and type IV collagens, laminin and fibronectin requires multiple domains of maspin and is initiated by maspin binding to a cell surface molecule. The major goal of this project is to identify the regions of the maspin molecule required for its biological activity and to characterize maspin binding partner(s) on corneal stromal fibroblasts. The aims of this proposal are the following: 1) To determine which regions of maspin are required for the induction of increased adhesion of cultured human stromal fibroblasts to ECM molecules. The functional maspin domains will be explored using rMaspin-ovalbumin hybrid proteins, site-specific maspin mutants, peptides and/or antibodies to specific maspin epitopes. 2) To characterize maspin binding to human corneal stromal fibroblasts and selected ECM molecules and determine which region(s) of maspin is required for binding. Maspin interaction with corneal stromal cells and selected ECM molecules will be characterized and the maspin domains determined by focusing on the loss-of-function maspin-ovalbumin hybrids and the results confirmed using maspin binding site peptides. 3) To identify and characterize the maspin-binding molecule(s) on corneal stromal fibroblast membranes. Maspin binding proteins(s) will be isolated by maspin cross-linking and their proteolytic peptides identified by MALDI-TOF analysis and/or N-terminal sequence analysis. They will be cloned, expressed in a yeast system and their interactions with maspin characterized using pull down assays and cross-linking studies.

描述（由申请人提供）：bMaspin 是丝氨酸蛋白酶抑制剂超家族的成员，主要研究其在癌细胞中的肿瘤抑制功能。像许多上皮细胞一样，正常角膜上皮和内皮细胞合成maspin。令人惊讶的是，正常人角膜基质细胞也合成maspin，这是已知合成这种分子的唯一非上皮细胞类型。在培养中，当传代的角膜基质细胞发展出类似于伤口成纤维细胞的特性时，它们会失去合成maspin的能力，这让人想起转移性癌细胞的丧失。然而，角膜基质成纤维细胞通过增加对 I 型和 IV 型胶原、纤连蛋白和层粘连蛋白的粘附来对外源性 maspin 治疗做出反应。研究 maspin 在角膜中的功能尤为重要，因为它调节基质附着和细胞运动的能力可能在调节基质伤口愈合和预防肿瘤发生和血管生成等事件中发挥关键作用。该提案特别提出了这样的假设：角膜基质成纤维细胞对 I 型和 IV 型胶原、层粘连蛋白和纤连蛋白的粘附力增加需要 maspin 的多个结构域，并且是由 maspin 与细胞表面分子结合引发的。该项目的主要目标是确定 maspin 分子生物活性所需的区域，并表征角膜基质成纤维细胞上的 maspin 结合伴侣。该提案的目的如下： 1) 确定 maspin 的哪些区域需要诱导培养的人基质成纤维细胞与 ECM 分子的粘附增加。将使用 rMaspin-卵清蛋白杂合蛋白、位点特异性 maspin 突变体、针对特定 maspin 表位的肽和/或抗体来探索功能性 maspin 结构域。 2) 表征 maspin 与人角膜基质成纤维细胞和选定的 ECM 分子的结合，并确定 maspin 的哪些区域需要结合。将表征 Maspin 与角膜基质细胞和选定的 ECM 分子的相互作用，并通过关注功能丧失的 maspin-卵清蛋白杂合体来确定 maspin 结构域，并使用 maspin 结合位点肽确认结果。 3) 鉴定和表征角膜基质成纤维细胞膜上的maspin结合分子。 Maspin结合蛋白将通过maspin交联进行分离，并通过MALDI-TOF分析和/或N端序列分析来鉴定其蛋白水解肽。它们将被克隆，在酵母系统中表达，并使用下拉分析和交联研究来表征它们与 maspin 的相互作用。