ANALYSIS OF THE AIRWAY ANTIPROTEINASE DEFENSE SYSTEM

（改编自申请人的摘要）蛋白酶在蛋白质合成中起主要作用。 炎症性肺病如囊性纤维化，哮喘， 慢性支气管炎、肺气肿和早产儿慢性肺病。一 更好地理解抑制剂对蛋白酶的调节 肺本身合成的蛋白质可以促进开发特异性 治疗这些疾病。 鳞状细胞癌抗原（SCCA）1和2是高表达的 分子量丝氨酸蛋白酶抑制剂（Serpin）家族。尽管SCCA 1 抑制溶酶体半胱氨酸蛋白酶、组织蛋白酶（cat）L、S和K，而 SCCA 2抑制糜蛋白酶样丝氨酸蛋白酶、catG和肥大细胞 糜酶SCCA 1和SCCA 2显示出组织限制性表达模式， 共同定位于气管、支气管和细支气管上皮。在 此外，SCCA 1的靶蛋白酶catS和catK由 气道上皮细胞这些强效弹性蛋白溶解半胱氨酸的另一个来源 肺中的蛋白酶是肺泡巨噬细胞。基于体外 SCCA 1和SCCA 2的抑制特性和分布模式 研究人员假设，这两种丝氨酸蛋白酶抑制剂可以保护 气道对抗蛋白酶介导的损伤。本提案的目的是 为了使用体外细胞培养和体内转基因来检验这一假设， 动物模型拟议项目的具体目标是：1） 表征外源性和内源性半胱氨酸的有害作用 蛋白酶catS和catK在体外支气管上皮细胞上的作用，2）测定 SCCA 1和/或SCCA 2是否能保护培养的支气管上皮细胞 从蛋白酶介导的损伤中分离靶蛋白酶，3） 确定SCCA 1和SCCA 2的靶向表达是否可以保护 蛋白酶介导的损伤。实验设计包括使用 与稳定转染结合以过表达SCCA 1 SCCA 2将通过以下方法研究气道上皮的屏障功能： 渗透性和跨上皮电阻测量以及结构 分析.亲和层析和免疫共沉淀将用于 鉴定SCCA 1和SCCA 2的靶蛋白酶， 体内损伤。将使用大鼠clara细胞10 kD蛋白启动子（CC 10） 将SCCA 1和SCCA 2基因的表达靶向肺。动物将 检查以确定对蛋白酶介导的 暴露于丙烯醛后的肺损伤这些研究将提高我们的 了解蛋白酶介导的损伤机制， 局部合成的抑制剂如SCCA 1和SCCA 2可以预防这种类型的 的损害。

(Adapted from applicant's abstract) Proteinases play a major role in the development of inflammatory lung diseases such as cystic fibrosis, asthma, chronic bronchitis, emphysema and chronic lung disease of prematurity. A better understanding of the regulation of proteinases by inhibitors synthesized by the lung itself could facilitate efforts to develop specific treatments for these diseases. Squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the high molecular weight serine proteinase inhibitor (serpin) family. Although SCCA1 inhibits lysosomal cysteine proteinases, cathepsins (cat)L,S and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, catG and mast cell chymase. SCCA1 and SCCA2 show a tissue restricted expression pattern and are co-localized in the tracheal, bronchial and bronchiolar epithelium. In addition, target proteinases of SCCA1, catS and catK, are expressed by the airway epithelial cells. Another source of these potent elastolytic cysteine proteinases in the lung is alveolar macrophages. Based on the in vitro inhibitory profiles and distribution patterns of SCCA1 and SCCA2 in the airways, the investigators hypothesize that these two serpins protect the airways against proteinase mediated injury. The objective of this proposal is to test this hypothesis using in vitro cell culture and in vivo transgenic animal models. The specific aims of the proposed project are to: 1) characterize the deleterious effects of exogenous and endogenous cysteine proteinases catS and catK on bronchial epithelial cells in vitro, 2) determine whether SCCA1 and/or SCCA2 can protect cultured bronchial epithelial cells from the proteinase-mediated injury and isolate the target proteinases, 3) determine whether targeted expression of SCCA1 and SCCA2 can protect the airways from proteinase-mediated injury. The experimental design involves use of cell cultures in conjunction with stable transfections to overexpress SCCA1 and SCCA2. Barrier function of the airway epithelium will be studied by permeability and transepithelial resistance measurements as well as structural analysis. Affinity chromatography and co-immunoprecipation will be used to identify the target proteinases of SCCA1 and SCCA2 in proteinase-mediated injury in vivo. The rat clara cell 10kD protein promoter (CC10) will be used to target expression of SCCA1 and SCCA2 genes to the lung. Animals will be examined to determine the extent of protection against proteinase-mediated lung injury following exposure to acrolein. These studies should enhance our understanding of the mechanisms of proteinase mediated injury and whether the locally synthesized inhibitors such as SCCA1 and SCCA2 can prevent this type of damage.