降钙素基因相关肽调控气管上皮多潜能干细胞分化和增殖机理的研究

Airway submucosal glands (SMGs) in the conducting airways play important roles in protection of the mucosal surface of the airway from inhaled pathogens. These structures secrete a large number of factors (e.g., lysozyme, lactoferrin, mucins, etc) into the airways that are involved in innate immunity and provide the bulk of mucus and fluid that coats the airways. Recent studies show SMGs also harbor nucleotide label-retaining cells (LRCs), suggesting their potential importance as a stem cell niche of the proximal airway. Given the fact that constantly recurrent injury in chronic airway diseases leads to malfunctional SMGs secretion, uncovering SMGs function at steady state and injury state is important to understand airway disease progression. Several hypotheses regarding functions of SMGs in airway repairing processes have emerged the recent years. One hypothesis is that neuronal inputs can regulate glandular secretion and promote stem cell proliferation or differentiation. Indeed, results from our previous studies demonstrated that 1) acute damages of airway epithelium leads to a transient induction of CGRP expression in SMGs, which signals glandular LRC progenitors to enter the cell cycle and produce transient amplifying cells (TAs) to affect airway repair. Also 2) chronic airway damages leads to persistent induction of CGRP as sustained mitogenic signals in the glandular LRC progenitor niche and this dysregulation of CGRP causes adaptive LRC niche formation in the surface airway epithelium (SAE). In this light, this proposal will focus on delineating regulatory mechanisms that control CGRP-dependent actions on airway stem cells. We hypothesize that the activation of Trpv1+ nerves promotes CGRP secretion indirectly activating stem cells proliferation and differentiation through secreting an unknown mitogen from SMGs. This hypothesis will be interrogated using GC-MS system to identify CGRP-responsive secretory factors and Trpv1 knockout mouse models that are capable of modulating airway injury with lacking of CGRP secretion. Most importantly, this proposal will use Rosa26-rtTA/TRE-H2B-EGFP transgenic mice to enrich and evaluate viable LRCs. Those single-cell suspended LRCs will be cultured in a novel ROCK inhibitor mediated airway liquid interface (ALI) culture system, which allow us to monitor LRCs differentiation and proliferation under stimulation of various secretory factors. Ultimately, this work attempt to answer two major questions: 1) How and why airway stem cells can response to induced CGRP during injury. 2) Is CGRP necessary for proper airway repairing after airway injury? We believe results from the proposed experiments will further define the regulatory machines of CGRP-dependent stem niche function and may aid in the development of new therapeutic approaches to treat chronic airway diseases.

呼吸道系统疾病发病率日趋上升。慢性的呼吸道疾病变会影响气管粘膜下分泌腺(SMGs: submucosal glands)的功能。SMGs分泌的黏液不仅是先天性免疫的重要组成部分，同时黏液含有的蛋白对维持呼吸道干细胞的多潜能性有必要作用。因此，SMGs功能紊乱又加剧了呼吸道病变的进程。前期研究表明，在损伤病变的呼吸道中，降钙素基因相关肽(CGRP)的分泌明显增加，导致SMGs干细胞微环境的失调，刺激呼吸道上皮增殖分化。但是CGRP刺激干细胞增殖分化的分子机制至今仍未阐明。本项目拟1）研究在CGRP的刺激下，SMGs黏液蛋白成份构成的变化。2）利用分离纯化活性标的记保留细胞(LRC) 研究在CGRP刺激下LRC的分化增殖规律。3)研究在CGRP分泌不足的情况下，LRC分化增殖功能的变化。以期研究结果能更深入地认识呼吸道干细胞的调控机制，并为治疗慢性呼吸道疾病提供有临床意义的用药靶点。

呼吸道分泌腺为呼吸道多潜能干细胞提供了良好的微环境(niches)。目前学术界对于呼吸道干细胞的调控机制没有充分阐述。主要的困难体现在缺少有效的实验手段对气管干细胞进行分离和纯化。并建立有效的体外培养方法对其增殖和分化过程进行研究评价。本课题研究首先有效的建立了以Rosa26-rtTA, tetON:H2B-GFP 转基因小鼠为实验对象的呼吸道损伤修复模型，同时采用流式细胞仪，组织荧光多重染色，建立体外Air liquid interface culture (ALI)二维培养和体外3D悬浮细胞培养体系，等多种实验方法，首次发现 1）H2B-GFP标记保留细胞具有多潜能性和较高的增殖性。2）首次成功分离纯化出具有活性的呼吸道标记保留细胞；3）首次用两种不同的体外培养体系里，分别验证了呼吸道标记保留细胞具有分化功能。因此证实H2B-GFP标记保留细胞是呼吸道多潜能干细胞。此外，我们还验证了TRPV1-CGRP调控通路在呼吸道细胞损伤修复过程中的功能。首次验证了CGRP 是刺激呼吸道细胞的增殖，有效的促进呼吸道的修复功能的关键分泌蛋白。TRPV1基因敲除小鼠呼吸道分泌腺缺失了CGPR的分泌功能，因此显著的延缓了呼吸道的损伤后修复时间，特别是导致呼吸道Clara细胞的缺失。