KCa3.1通道对变应性鼻炎Tfh细胞免疫调控作用及机制研究

It has been shown that Th1/Th2 cells imbalance was involved in the immunopathology of allergic rhinitis.A newly-discovered Tfh cell has been proved to play the most critical role in assisting B cell to produce antibody and promote humoral immune response.Our pre-experiment showed that Tfh cells proportion was elevated in the peripheral blood of allergic rhinitis patients ,indicating that Tfh cells may play a role in the allergci rhinitis.Our earlier work demonstrated that a kind of calcium channel calling Orail channel which promoted the inflow of extracellular calcium ions was elevated in the nasal mucosa, indicating that inflow of extracellular calcium ions would lead to exacerbation of inflamation. When Orail channel was blocked by shRNA method,inflammation-associated cytokines in allergic rhinitis mice were obviously reduced. Our pre-experiment showed that a kind of potassium channel calling KCa3.1 channel which promoted the outflow of intracellular potassium ions and inflow of extracellular calcium ions in the immune reaction was elevated in the nasal mucosa of allergic rhinitis patients and allergic rhinitis mice.In this study, we would further investigate the role of KCa3.1 channels for Tfh cells immunoregulation in allergic rhinitis，we speculate that ions alteration may activate NF-κB signaling pathway, leading to the activation of Tfh cells and secretion of inflammatory factors, and then basophils were recruited and degranulated.This project would will be of great importance in illustrateing deeply the pathogenesis of allergic rhinitis and exploring gene therapy method.

既往研究认为，Thl/Th2细胞间的免疫反应失衡是变应性鼻炎的免疫学基础。近年来新发现的Tfh细胞证实是辅助B细胞产生抗体及体液免疫应答最主要和最关键的T细胞亚群。本项目前期预实验发现Tfh细胞在变应性鼻炎患者外周血中比例升高，说明Tfh细胞对变应性鼻炎具有一定作用。我们前期工作证明钙离子Orail通道在变应性鼻炎小鼠鼻黏膜中表达升高，进而促进细胞外钙离子内流导致炎症进展，采用shRNA技术下调Orail表达后，小鼠炎症相关因子降低。本项目预实验发现钾离子KCa3.1通道在变应性鼻炎患者及小鼠鼻黏膜中表达升高，进而促进钾离子外流协同钙离子内流而参与免疫反应。本项目将进一步研究KCa3.1通道对Tfh细胞免疫调控作用，设想离子改变可激活NF-κB信号通路，促进Tfh细胞活化、分泌炎症因子，影响嗜碱性粒细胞而出现募集、脱颗粒。这些研究对深入阐明变应性鼻炎发病机制及探索基因治疗具有重要意义。

KCa3.1通道是一种位于细胞膜上的钾离子通道，可介导钾离子外流及钙离子内流。我们通过构建变应性鼻炎(Allergic rhinitis, AR)小鼠模型，检测研究KCa3.1通道在AR小鼠模型中的表达、作用及KCa3.1通道选择性阻断剂TRAM-34干预作用，结果发现：KCa3.1通道在AR小鼠鼻黏膜组织中表达明显增高，KCa3.1通道在AR发病过程中具有一定作用，TRAM-34干预可阻断KCa3.1通道，然后引起钾离子外流及钙离子内流减少，从而有效地减轻小鼠AR炎症及发作症状。我们进一步研究了慢病毒Lenti-KCa3.1-shRNA对AR小鼠模型的干预作用及机制，结果显示：Lenti-KCa3.1-shRNA干预可阻断KCa3.1通道，然后引起钾离子外流及钙离子内流减少，炎症减轻，免疫反应抑制。接着我们深入进行AR小鼠模型KCa3.1通道对Tfh细胞免疫调控机制研究，结果显示: AR小鼠KCa3.1通道对Tfh细胞具有免疫调控作用，可能是通过NF-κB信号通路激活Tfh细胞，促进IL-21分泌，采用慢病毒Lenti-KCa3.1-shRNA干预通过影响NF-κB信号通路导致Tfh细胞活性下降，IL-21分泌降低。接着，我们分离、培养及鉴定了小鼠鼻腔黏膜中的外胚层间充质干细胞(ectomesenchymal stromal cells, ECTO-MSCs)， 观察其对AR小鼠的干预及机制，结果发现：小鼠鼻黏膜来源ECTO-MSCs能迁徙到AR小鼠的鼻黏膜炎症部位中。注射ECTO-MSCs能显著改善小鼠的AR症状，减少鼻黏膜中嗜酸性粒细胞浸润程度，减轻鼻黏膜变应性炎症。最后我们还进行了细胞因子信号抑制蛋白3 (suppressor of cytokine signaling 3，SOCS3)介导间充质干细胞调节免疫反应的研究，结果发现：IFN-γ能显著提高MSCs中SOCS3的表达水平，MSCs经IFN-γ活化后能显著抑制T细胞和B细胞(尤其是B细胞)的增殖。SOCS3可能通过细胞-细胞接触的途径，介导了MSCs对淋巴细胞的抑制作用。项目资助发表SCI论文6篇。培养博士生6名，其中5名已经取得博士学位，2名在读。项目投入经费730000.00元，支出620394.50元，各项支出基本与预算相符。剩余经费109605.50元，剩余经费计划用于本项目研究后续支出。