气道内高压诱导气道重塑的信号链接机制

The existence of cross-linking points between the biological effects generated by the process of mechanical stress stimuli affecting on target cells and classic mechanism of airway remodeling in chronic airway inflammatory diseases, and the presention of abnormal high pressure in small airway of chronic airway inflammatory diseases, these evidences lead us to speculate that the mechanical stress induced by high pressure in small airway may play important role in the process of airway remodeling . TRP channels located on the membrane can perceive mechanical stress and thus translate this mechanical signal into biological signal. The relationships between the levels of TRPs and the extent of airway remodeling will be clarified by animal airway remodeling models and simulation of high airway pressure to intervene cultured target cells in vitro. The heterogeneity TRPs involved in airway remodeling will be screened and the mechanisms related to this process will be explored by means of TRPs gene transcription interference and specific inhibitor. The cis- acting elements, sites and trans-acting factors such as SP1, CREB, GR will be analyze by constructing a series of transient expression vector of TRPs gene promoter and the key regulatory elements will be identified by way of construction the TRPs luciferase reporter gene plasmid containing of site-directed mutagenesis of promoter regulatory elements. The purpose of this study is to confirm the role of chronic airway inflammatory stimuli and corticosteroids in the TRPs gene transcriptional activation and transcriptional repressor. The clarification of mechanisms of TRP channels in airway remodeling induced by abnormally sustained high airway pressure may make TRP channels become a new target proteins in the prevention and treatment of airway remodeling.

机械应力刺激对靶细胞产生的生物学效应与气道重塑的经典机制存在相互交联点，而在慢性气道炎症性疾患的小气道压力常呈异常增高状态，由此产生的机械应力在气道重塑过程中扮演了什么角色？胞膜上感知机械应力刺激的TRP通道可能将此机械信号转化为生物学信号。本研究通过气道重塑动物模型及体外模拟气道内高压干预靶细胞，明确TRP表达水平及与气道重塑程度的相关性，并运用基因沉默技术及特异性抑制物筛选出与气道重塑相关的目的TRPs，初步探讨TRPs参与该过程的形式和机制。通过构建目的TRPs基因启动子系列的表达载体，分析可能存在的顺式调控元件及转录因子，进一步构建调控元件定点突变荧光素酶报告基因质粒来确定关键调控元件。试图证实慢性气道炎性刺激及糖皮质激素对目的TRPs基因的转录激活及转录阻遏调控作用。TRP通道在持续异常增高的气道内压力引起的气道重塑机制的进一步明确，可能使其成为预防和治疗气道重塑的新型靶蛋白。

项目背景：气道阻力增加及压力异常增高是多种慢性气道炎性疾病的普遍现象，并最终导致气道狭窄促成气流受限。其可能涉及牵张力及正压力激活细胞膜上的机械敏感性离子通道（MSC），促进胞外机械信号向胞内的转导，参与气道重塑的后续效应。研究内容：本研究通过通过搜集COPD患者组织标本、气道重塑动物模型及体外模拟气道内高压干预靶细胞，明确TRP表达水平及与气道重塑程度的相关性，初步探讨TRPs参与该过程的形式和机制。其继发效应是细胞内Ca2+浓度增高、炎性介质释放，进而引起平滑肌细胞增生肥大、细胞基质沉淀、杯状细胞增生肥大。重要结果：COPD患者组TRPC1、MMP9和TGFβ1表达水平升高。COPD患者组组织标本中TRPC1蛋白主要分布于支气管上皮细胞腔内侧，表达于柱状细胞或杯状细胞胞浆内；Image-Pro图像软件分析结果示，卵蛋白刺激组的豚鼠显著表现出支气管壁增厚、基底膜胶原沉积、IL-5和IL-13炎症因子增加、嗜酸性粒细胞（Eos）占总细胞数的百分比增加等病理变化。采用柔性基底加载装置（FX-4000T）牵拉16HBE细胞，加载时间分别为：0.5 h, 1 h, 1.5 h, 2 h。发现与空白对照组相比，4个时间段TGFβ1、FGF-2、IL-13、MMP9和Ca2+的表达均明显高于空白对照组，在0.5 h时其增幅最大。选择0.5 h机械牵拉组，以TRPC1抑制剂SKF96365、PKC抑制剂HA-100干预，其TGFβ1、FGF-2和Ca2+的表达均较0.5 h机械牵拉组明显降低。当给予TRPC1 siRNA、钙离子通道阻滞BAPTA-AM干预后，IL-13、MMP9的表达明显降低。关键数据：COPD患者组组织标本中TRPC1蛋白主要分布于支气管上皮细胞腔内侧。在动物实验中，卵蛋白刺激组的豚鼠显著表现出支气管壁增厚、IL-5和IL-13炎症因子增加、嗜酸性粒细胞（Eos）占总细胞数的百分比增加。在细胞实验中，牵拉16HBE细胞，发现在0.5 h时，TGFβ1、FGF-2、IL-13、MMP9和Ca2+的表达均明显高于空白对照组。以SKF96365、HA-100、TRPC1 siRNA、BAPTA-AM干预，上述因子的表达明显降低。科学意义：TRPC1通道在持续异常增高的气道内压力引起的气道重塑机制的进一步明确，可能使其成为预防和治疗气道重塑的新型靶蛋白。