Repair of Airway Epithelium Following Chlorine Lung Injury

描述(由申请人提供)：急性高水平暴露于损害呼吸道的化学物质可导致危及生命的肺损伤。氯气是一种剧毒的呼吸道刺激物，被认为是一种化学威胁物质，因为它可能会在工业事故或恐怖袭击中释放出来。像许多肺部毒物一样，氯也会对呼吸道衬里的上皮细胞造成伤害。呼吸道上皮细胞屏障的丧失会导致急性肺损伤的其他后遗症，包括肺水肿、呼吸道阻塞、炎症和呼吸道感染。有效的呼吸道上皮修复和屏障功能的恢复是解决氯性肺损伤的关键。呼吸道上皮在损伤后通过干细胞的增殖和分化得到修复。参与呼吸道上皮修复的一种细胞是基底细胞。低亲和力神经生长因子受体(NGFR)已被确定为基底细胞和其他组织中的上皮干细胞的标志物，但该蛋白在干细胞功能中的作用尚不清楚。NGFR可与多种神经营养因子配体结合，包括神经生长因子(NGF)、脑源性神经营养因子和神经营养因子3。本研究假设NGFR控制氯致肺损伤后肺上皮干细胞的增殖、存活和分化。在特定目标1中，将从正常小鼠和缺乏NGFR功能的突变小鼠的气管中纯化基底细胞。培养细胞的存活、增殖和分化为其他类型的上皮细胞(纤毛细胞和Clara细胞)的能力将在有无NGFR配体的情况下进行测量。在特定目标2中，根据首席调查员实验室制定的方案，野生型、NGFR基因敲除和NGF转基因小鼠将暴露在氯气中。神经营养因子的效果将通过鼻腔输送配体或使用先前在首席研究人员实验室开发的过表达NGF的转基因小鼠来测试。在氯暴露后的不同时间将测量气管上皮修复的程度。在具体目标3中，将研究神经营养因子在氯气暴露后兔上皮修复中的作用。兔子将被用于这些研究，因为在这个物种中，基底细胞在呼吸道中的分布更广泛，这与人类的更相似。拟议中的实验不仅将研究上皮修复的机制，还将促进治疗剂的开发，这些治疗剂可用于刺激吸入有毒化学品后肺损伤的缓解。 公共卫生相关性：本申请是响应PAR-10-019，抵消转化研究中的探索性/发展性项目(R21)而提交的。拟议的实验旨在了解吸入有毒气体后肺部如何自我修复，目的是开发基于刺激肺细胞加速修复的肺损伤治疗方法。这类研究是通过资金机会公告寻求的，因为人们担心美国平民可能会受到在恐怖袭击中故意释放的剧毒化学品或在工业事故或自然灾害中无意释放的高毒化学品的不利影响。

DESCRIPTION (provided by applicant): Acute high-level exposures to chemicals that damage the respiratory tract can cause life-threatening lung injury. Chlorine gas is a highly toxic respiratory irritant that is considered a chemical threat agent because of the possibility that it could be released in industrial accidents or terrorist attacks. Chlorine, like many pulmonary toxicants, produces injury to epithelial cells lining the respiratory tract. Loss of the respiratory epithelial cell barrier contributes to other sequelae observed in acute lung injury, including pulmonary edema, airway obstruction, inflammation, and respiratory infections. Efficient repair of respiratory epithelium and restoration of barrier function are essential for resolution of chlorine-induced lung injury. The epithelium of the respiratory tract is repaired following injury through the proliferation and differentiation of stem cells. One type of cell involved in the repair of airway epithelium is the basal cell. The low-affinity nerve growth factor receptor (NGFR) has been identified as a marker for basal cells, as well as for epithelial stem cells in other tissues, but the role of this protein in stem cell function is unknown. NGFR binds multiple neurotrophin ligands, including nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3. The hypothesis to be tested in the proposed studies is that NGFR controls the proliferation, survival, and differentiation of lung epithelial stem cells following chlorine-induced lung injury. In Specific Aim 1, basal cells will be purified from the trachea of normal mice and from mutant mice lacking functional NGFR. The ability of the cultured cells to survive, proliferate, and differentiate into other types of epithelial cells (ciliated cells and Clara cells) will be measured in the presence or absence of NGFR ligands. In Specific Aim 2, wild-type, NGFR knockout, and NGF transgenic mice will be exposed to chlorine gas according to a protocol established in the Principal Investigator's laboratory. Effects of neurotrophins will be tested by delivering ligands intranasally or by using NGF-overexpressing transgenic mice previously developed in the Principal Investigator's laboratory. The extent of tracheal epithelial repair will be measured at various times after chlorine exposure. In Specific Aim 3, the effect of neurotrophins on epithelial repair following chlorine exposure in rabbits will be investigated. Rabbits will be used for these studies because of the wider distribution of basal cells in the airways in this species which is more similar to that in humans. The proposed experiments will not only investigate mechanisms of epithelial repair, but will also foster the development of therapeutic agents that could be used to stimulate resolution of lung injury following inhalation of toxic chemicals. PUBLIC HEALTH RELEVANCE: This application is submitted in response to PAR-10-019, CounterACT Exploratory/Developmental Projects in Translational Research (R21). The proposed experiments are designed to understand how the lung repairs itself after inhalation of a toxic gas with the goal of developing therapy for lung injury based on stimulating lungs cells to accelerate repair. This type of research is sought through the Funding Opportunity Announcement because of concerns that U. S. civilians could be adversely affected by highly toxic chemicals released intentionally in terrorist attacks or unintentionally in industrial accidents or natural disasters.