整合素-β1对哮喘气道平滑肌表型转化和钙通道功能的双重调控作用及干预研究

Asthma is characterized by reversible airway inflammation, airway remodeling, and airway hyperresponsiveness(AHR). Although our understanding of its pathophysiological mechanisms continues to evolve, the relative contributions of airway inflammation are still debated. Asthma was recognized as an inflammatory disease, with chronic inflammation inducing airway remodeling and AHR. However, persistence of airway dysfunction despite inflammatory control is observed in chronic severe asthma of both adults and children. More recently, the central roles for phenotype transformation and calcium channels' functions of airway smooth muscle cells (ASMC) are emerging, with airway dysfunction often resulting in airway remodeling and AHR, suggesting that ASMC plays a crucial part in the pathogenesis of asthma. It is noteworthy that the disorder of signal transduction system is critical for the development of ASMC's pathophysiology involve the processes of phenotype transformation and calcium channels' functions. In addition, a central role for integrin-β1 in the pathogenesis of airway remodeling and AHR has been explored by our preliminary experiments, highlighting it may function as key regulator of phenotype modulation and calcium channels' function of ASMC. However, the regulating roles of integrin-β1 in ASMC and its mechanisms have not been reported. So we plan to carry out this research to explore the effect of the endogenous integrin-β1on phenotype transformation and the calcium channels' functions of ASMC in asthma. Firstly, we perform several experiments on the chronic asthma model of mice both in vitro and in vivo assays, and on the clinical cases. The purpose of these experiments is to verify our scientific hypotheses that integrin-β1 can regulate both the phenotype transformation and the calcium channels' functions of ASMC in asthma. Secondly, the recombinant lentivirus vectors are constructed to block or promote the gene expression of integrin-β1. Then many biotechnologies are used to evaluate the effects of integrin-β1 on phenotype transformation, calcium channels' proteins, membrane potential, intracellular calcium concentration of ASMC, and to explore the signal transduction mechanisms from cellular, protein and genetic levels. These assays rely on a suite of technologies including patch clamp, protein chip, transfection in vitro and in vivo, genetic intervention, immuno-electrophysiology, etc. Lastly, the exogenous recombinant lentivirus vectors are used to disturb the endogenous integrin-β1 gene's expression both in vivo and in vitro assays, in order to suppress the phenotype transformation of ASMC, attenuate the calcium influx, alleviate the airway remodeling, and decrease AHR in asthma. All the above experiments will demonstrate that integrin-β1 is the key regulator of phenotype transformation and calcium channels' functions of ASMC, and suggest that it may be a potent novel target for the future development of genetic intervention of asthma.

气道平滑肌细胞（ASMC）表型转化及钙通道功能异常是导致哮喘气道重塑和气道高反应性（AHR）的中心枢纽及关键环节，但其机制尚不清楚。本项目拟在我们前期工作基础上，开展小鼠哮喘模型在体、离体实验及临床研究，确认内源性整合素（Integrin）-β1对哮喘ASMC表型转化及钙通道功能异常的双重调控作用；在此基础上，用构建重组慢病毒载体的方法使Integrin-β1基因表达沉默或过度表达，采用膜片钳、蛋白质芯片、体内外转染及免疫电生理等技术，从细胞、蛋白、基因不同层面及不同胞外环境，研究其对哮喘ASMC表型转化、钙通道蛋白、膜电位、胞内钙离子浓度的影响及其信号转导机制；同时，利用人工构建的外源性shRNA重组慢病毒载体的基因靶向干预作用，抑制内源性Integrin-β1基因表达，达到调控ASMC表型转化、钙通道功能、改善气道重塑、AHR的目的，为探寻哮喘发病机制及后续基因干预治疗提供理论依据。

气道重塑是支气管哮喘（简称哮喘）的一个重要病理特征，但其具体发生机制尚不明确。Integrin-β1广泛存在于气道平滑肌细胞（ASMC）表面，并能调控ASMC 多个重要的生理过程，故可能对哮喘气喘重塑有重要影响。为了更好更全面的了解Integrin-β1在哮喘中的作用机制，本研究首先检测了Integrin-β1在小鼠哮喘模型中的表达，发现在哮喘组中Integrin-β1的表达显著升高；接着应用 RNA 干扰技术建立小鼠ASMC细胞的Integrin-β1基因缺陷细胞株，RT-PCR及 western blot检测显示最终选定的缺陷细胞株干扰效率效果显著（P<0.05）；进一步利用流式细胞术检测了Integrin-β1 shRNA表达载体对小鼠ASMC细胞周期、增殖与凋亡的影响，结果显示Integrin-β1缺陷能有效抑制小鼠ASMC增殖，将哮喘ASMC阻滞在G0/G1期，从而有效抑制其细胞内 DNA合成，并能有效诱导哮喘ASMC凋亡细胞；然后利用Elisa检测细胞炎症因子IL-6与IL-8的表达，shRNA表达载体能有效抑制哮喘ASMC分泌IL-6和IL-8；同时RT-PCR结果提示该shRNA 表达载体能有效下调哮喘ASMC内CyclinD1及c-Fos mRNA 表达，western blot结果显示shRNA表达载体不会影响哮喘ASMC内ERK1/2 和STAT6的总蛋白表达，但能降低ASMC内p-ERK1/2、CyclinD1及C-fos蛋白表达、提高p-STAT6 蛋白表达；在钙通道功能研究中，结果显示哮喘组小鼠肺组织的STIM1、Orai1、TRPC1、NFAT2 mRNA表达量比正常组小鼠明显增高，经特异性Integrin-β1 shRNA靶向干扰后，干预处理组的STIM1 mRNA水平与空白对照组、阴性对照组、阳性对照组之间表达量无显著性差异，但TRPC1、Orai1、NFAT2 mRNA水平显著低于空白对照组、阴性对照组及阳性对照组。