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MACROPHAGE NEUTRAL PROTEINASES

巨噬细胞中性蛋白酶

基本信息

批准号：
3351484
负责人：
CARLO L. MAINARDI
金额：
$ 15.08万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-09-01 至 1990-06-30
项目状态：
已结题

项目摘要

Macrophages are frequently found at sites of chronic inflammatory conditions which are associated with tissue destruction and fibrosis. Despite this association, little is known about the enzymatic constituents of macrophages which might mediate the degradation of the extracellular matrix components. Previous studies have indicated that these cells secrete several different proteinases which are capable of degrading the collagenous components of the matrix in a type-specific manner. The model used in this proposal is the "in vivo" activated rabbit alveolar macrophage. The first objective of this proposal is to purify and characterize the collagenolytic proteinases secreted by these cells. Included are: 1) a collagenase which specifically degrades native type (pericellular) V collagen; 2) an interstitial collagenase; and 3) an enzyme which degrades type IV (basement membrane) collagen. These enzymes will be purified using standard methods of chromatography and characterized by their molecular composition, substrate specificity, mechanisms of activation, and inhibition by proteinase inhibitors. Monospecific polyvalent antibodies will be raised against these enzymes. The cleavage products of the collagens which are generated by the action of these enzymes will be characterized. Initial studies will focus on the degradation of type V collagen by the specific macrophage proteinase and the degradation of type IV collagen by the neutrophil enzyme, elastase. Methods employed will include peptide mapping of collagen fragments isolated by polyacrylamide gel electrophoresis and by column chromatography as well as amino acid analysis and amino terminal sequencing of fragments isolated by the latter method. The regulation of synthesis and release of these enzymes will be investigated. The effect of various modulating agents (corticosteroids, cyclo-oxygenase inhibitors, bacterial products, etc.) will be determined in vitro by incubating the cells with the various agents, and in vivo by administering the agents to the animals. Enzyme secretion will be quantitated by assay of enzyme activity and by immunochemical techniques. These studies will contribute to our understanding of the role of macrophages in the destructive and fibrotic pulmonary diseases.

巨噬细胞常出现在慢性炎症部位，与组织破坏和纤维化相关的病症。尽管有这种联系，但对酶的成分知之甚少。可能介导细胞外基质降解的巨噬细胞基质成分以前的研究表明，这些细胞分泌几种不同的蛋白酶，能够降解以类型特异性方式抑制基质的胶原成分。模型在该建议中使用的是“体内”活化的兔肺泡巨噬细胞。该提案的第一个目标是纯化和表征由这些细胞分泌的胶原蛋白溶解蛋白酶。包括：（1）a 特异性降解天然型（细胞周）V的胶原酶胶原; 2）间质胶原酶;和3）降解胶原的酶 IV型（基底膜）胶原蛋白。这些酶将被纯化，色谱标准方法，并通过其分子特征组成、底物特异性、激活机制，以及蛋白酶抑制剂的抑制作用。单特异性多价抗体会对这些酶产生对抗胶原蛋白的裂解产物是由以下作用产生的：这些酶将被表征。初步研究将侧重于通过特异性巨噬细胞蛋白酶降解V型胶原， IV型胶原蛋白被中性粒细胞酶弹性蛋白酶降解。所采用的方法将包括胶原蛋白片段的肽图谱通过聚丙烯酰胺凝胶电泳和柱色谱分离以及片段的氨基酸分析和氨基末端测序用后一种方法分离。这些酶的合成和释放的调节将是研究了各种调节剂（皮质类固醇，环氧合酶抑制剂、细菌产物等）将确定在体外通过将细胞与各种试剂一起孵育，在体内通过将所述试剂施用给动物。酶分泌将是通过酶活性测定和免疫化学技术定量。这些研究将有助于我们了解巨噬细胞在破坏性和纤维化肺部疾病中的作用。