瞬时感受器电位M7与TGF-β1/Smad信号通路交互影响调控哮喘气道重塑的研究

We have confirmed that TGF-β1/Smad signalling pathway plays an important role in airway remodeling in asthma and found that transient receptor potential melastatin 7 （TRPM7）channel is high expression in primary airway smooth muscle cells (ASMCs) of asthmatic mice. The present research is designed to explore the potential mechanism of this TRPM7 upregulation using cell cultures, pharmacology experiments and molecular biological techniques. Then we seek to investigate the effects of TRPM7 overexpression/silence on the proliferation and apoptosis of ASMCs isolated from asthmatic mice as well as TGFβRⅠ/Ⅱand Smad2/3/7 expression after TGF-β1 stimulation. Dual-Luciferase Reporter Assay System will be applied to identify whether TRPM7 is a posttranscriptional target gene of Smad3. We will study TRPM7-protein interactions with the yeast two-hybrid system. Meanwhile, we intend to study the effects of TRPM7 overexpression/silence on airway remodeling and TGF-β1/Smad signal pathway in asthmatic mice. By this research we expect to elucidate a cross-talk between TRPM7 and TGF-β1/Smad in the pathogenesis of airway remodeling and provide evidence for TRPM7 as a potential therapeutic target in asthma.

我们已经证实TGF-β1/Smad信号通路参与调控支气管哮喘气道重塑，也发现气道重塑的哮喘小鼠原代气道平滑肌细胞（ASMCs）瞬时感受器电位M7（TRPM7）表达显著上调。本项目拟以细胞培养、药理学实验及分子生物学技术首次探讨哮喘小鼠气道平滑肌细胞TRPM7表达上调的可能机制；观察TGF-β1作用下哮喘小鼠气道平滑肌细胞增殖、凋亡及Smad2/3/7、TGFβRⅠ/Ⅱ的表达活化是否受TRPM7过表达/沉默的调控；以双荧光素酶报告基因验证Smad3是否靶向调控TRPM7；通过酵母双杂交实验寻找与TRPM7相互作用的蛋白质，探讨TRPM7发挥其生物学功能可能涉及的分子机制；同时动物模型观察TRPM7过表达/沉默对哮喘小鼠气道重塑及TGF-β1/Smad信号通路的作用。旨在获得TRPM7与TGF-β1/Smad交互影响调控哮喘气道重塑的证据，为确立TRPM7作为哮喘治疗的新靶标提供科学依据。

目的：我们已经证实TGF-β1/Smad信号通路参与调控支气管哮喘气道重塑，也发现瞬时感受器电位M7（TRPM7）在哮喘肥大细胞中起重要作用。本项目以细胞培养、药理学实验及分子生物学技术首次探讨哮喘小鼠气道平滑肌细胞TRPM7表达上调的可能机制。.方法：建立哮喘小鼠模型，以ELISA和western blotting检测TGF-β1和TRPM7。原代培养哮喘平滑肌细胞，用荧光实时定量PCR和western blotting检测TGF-β1刺激对平滑肌细胞内TRPM7表达的影响，钙荧光探针（indo-1 fluorescence)检测TRPM7敲除对哮喘小鼠气道平滑肌细胞内钙离子[Ca2+]i的影响。MTT法和流式细胞术检测TRPM7敲除对哮喘小鼠气道平滑肌细胞增殖凋亡的影响。染色质免疫共沉淀技术(ChIP)检测Smad3与TRPM7基因启动子区结合能力变化。.结果：与正常小鼠对照组相比，在哮喘小鼠组中，通过ELISA检测肺泡灌洗液(BALF)中TGF -β1表达水平升高，肺组织TRPM7的表达增加，与小鼠气道被OVA激发的时间呈正相关。在细胞实验中发现TGF-β1以浓度依赖性的方式诱导ASMCs中TRPM7 mRNA以及蛋白表达增加，促进细胞内钙离子浓度增加。通过SMAD3抑制剂SIS3以及TGFβR抑制剂LY21097761抑制Smad通路后，ASMCs中TRPM7 mRNA以及蛋白，胞内钙离子浓度较抑制前明显下降。基因敲除TRPM7抑制TGF-β1诱导的ASMCs增殖。染色质免疫共沉淀技术（CHIP）显示TGF-β1通过促进p-SMAD3与TRPM7启动子上游1454-1470bp区域的结合，增强TRPM7的转录，促进TRPM7的表达。.结论：TGF-β1通过TGFβR/Smad3调控TRPM7通道钙离子内流，促进气道平滑肌增殖。获得TRPM7与TGF-β1/Smad交互影响调控哮喘气道重塑的证据，为确立TRPM7作为哮喘治疗的新靶标提供科学依据。