ANALYSIS OF THE AIRWAY ANTIPROTEINASE DEFENSE SYSTEM

(摘自申请者的摘要)蛋白水解酶在 发展为炎症性肺部疾病，如囊性纤维化、哮喘、 慢性支气管炎、肺气肿、早产儿慢性肺部疾病。一个 进一步了解抑制剂对蛋白水解酶的调节作用 由肺本身合成的可以促进努力开发出特定的 这些疾病的治疗方法。 鳞状细胞癌抗原(SCCA)1和2是高 分子量丝氨酸蛋白酶抑制物(丝氨酸酶)家族。虽然SCCA1 抑制溶酶体半胱氨酸蛋白酶、组织蛋白(CAT)L、S和K，而 SCCA2抑制胰凝乳酶样丝氨酸蛋白酶、CATG和肥大细胞 凝乳酶。SCCA1和SCCA2呈现组织限制性表达模式 共定位于气管、支气管和细支气管腺上皮。在……里面 此外，SCCA1的靶蛋白CAT和CatK是由 呼吸道上皮细胞。这些有效的弹力分解半胱氨酸的另一个来源 肺中的蛋白水解酶是肺泡巨噬细胞。基于体外实验 SCCA1和SCCA2的抑制谱及在体内的分布 航空公司，调查人员假设这两条蛇保护着 呼吸道对抗蛋白酶介导的损伤。这项提议的目标是 使用体外细胞培养和体内转基因来验证这一假说 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 蛋白水解酶CAT和CatK对体外培养的支气管上皮细胞的影响 SCCA1和/或SCCA2对培养的支气管上皮细胞的保护作用 从蛋白水解酶介导的损伤中分离出目的蛋白，3) 确定SCCA1和SCCA2的靶向表达是否能保护 保护呼吸道免受蛋白水解酶的损伤。实验设计涉及到使用 细胞培养结合稳定的转染法过表达SCCA1 和SCCA2。呼吸道上皮屏障功能的研究将通过 通透性和跨上皮阻力测量以及结构 分析。亲和层析和免疫共沉淀将用于 确定SCCA1和SCCA2在蛋白水解酶中的靶蛋白 活体损伤。将使用大鼠Clara细胞10kD蛋白启动子(Cc10) 目的：将SCCA1和SCCA2基因靶向表达到肺组织。动物将会是 检查以确定对蛋白酶介导的保护程度 暴露于丙烯醛后的肺损伤。这些研究应该会增强我们的 了解蛋白水解酶介导的损伤的机制以及是否 局部合成的抑制剂，如SCCA1和SCCA2，可以防止这种类型 造成的损害。

(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.