气道炎症时神经肽调控肥大细胞脱颗粒的机制研究

Airway allergic diseases are closely related to inflammation. It is well-documented that the mast cell (MC) degranulation plays an important role in this inflammatory process. However, very little is known about the mechanism regarding the neural control of MC degranulation. Numerous studies have showed that the substance P(SP) released from terminals of sensory nerve can induce MC degranulation; nevertheless, the underlying mechanism is unclear. The purpose of this proposal is to explore a novel mechanism for neuropeptide-controlled MC degranulation. Based on our previous work, we propose that a "two-way" mechanism may be involved in the modulation of SP-induced MC degranulation. For non-immune degranulation, SP combined with neurokinin receptor (NK-R) modulates MC by inducing eosinophil (EOS). SP could also modulate the immune degranulation by influencing the expression of IgE receptor (FcεRI) on MC. To test our hypothesis, we will first develop a mice disease model. As a routine experiment procedure, the EOS will be isolated and purified, and the bone marrow-derived MC(BMMC) prepared. Furthermore, we will evaluate how the EOS stimulated by SP modulate the MC degranulation. By using gene silencing technic, two cell strains will be prepared, i.e., BMMCs with no expression of NK-R mRNA and no expression of FcεRImRNA corresponding to IgE receptor. Subsequently, MC degranulation as well as FcεRI expression of these two different BMMC strains to SP stimulus will be compared with wild type BMMC. In summary, these studies should provide us with better understanding of the mechanism of neuropeptide-controlled MC deranulation.

呼吸道变应性疾病的实质是炎症，其中肥大细胞（mast cell, MC）脱颗粒扮演重要角色。目前对MC脱颗粒的神经调控机制知之甚少。P物质（substance P,SP）能诱导MC脱颗粒，但机制不明。基于前期工作，我们假设呼吸道炎症时SP借助速激肽受体（NK-R）诱导嗜酸性细胞（eosinophil，EOS）调控MC实现非免疫性脱颗粒，同时通过影响MC表面IgE受体FcεRI调控免疫性脱颗粒，形成双重脱颗粒调控机制。我们将建立小鼠疾病模型，分离提纯EOS,制备骨髓肥大细胞（bone marrow-derived MC，BMMC)， 探讨SP诱导EOS调节MC脱颗粒机制；同时利用基因沉默技术分别制作不表达NK-R mRNA和不表达IgE受体FcεRImRNA的BMMC 细胞株，以野生型BMMC细胞株为对照，比较SP刺激对该细胞株脱颗粒及表达FcεRI的情况，以便阐明MC脱颗粒的一种新的机制。

变应性鼻炎（allergic rhinitis，AR）是一类由IgE/FcεRⅠα介导的以肥大细胞（mast cell,MC）脱颗粒为主的鼻黏膜免疫反应。AR发病率高达30%~40%，呈现逐年递增的趋势。而AR治疗的一线用药主要包括抗组胺、抗白三烯等MC脱颗粒阻滞药物，但其治疗效果仍是差强人意。究其原因是对MC脱颗粒的机制仍不十分清楚。因此有必要从另一个角度探究MC脱颗粒的新机制。. 研究发现鼻黏膜中MC分布与感觉神经相伴行，而神经末梢释放的神经肽P物质（substance P,SP）可能通过调控MC表面NK-1R和FcεRⅠα两种受体表达进而刺激MC脱颗粒。为此本课题通过在体外“模拟”气道炎症时的内环境，重点探究AR发病时SP调控肥大细胞脱颗粒的非免疫性机制——神经机制。. 处死小鼠后，提取股骨干细胞，用不同浓度IL-4进行体外培养。四周后收集各组细胞及上清液，进行甲苯胺蓝染色、流式细胞术检测CD117鉴定MC，流式细胞术以及Western Blotting分析不同组骨髓肥大细胞FcεRⅠα和NK-1R表达情况。经鉴定培养成功的骨髓肥大细胞中分别加入不同浓度SP孵育半小时，计算组胺释放率。以上结果初步证实IL-4参与诱导时可促使骨髓MC高表达FcεRⅠα和NK-1R，成功建立SP介导的MC脱颗粒模型。SP调控MC脱颗粒存在其特异受体NK-1R介导的非免疫性调控机制（神经机制）及FcεRⅠα介导的免疫性调控机制。 . 进一步观察发现AR发作呈现晨起或夜间加重的鼻高反应性，而MC脱颗粒释放组胺、白三烯等炎性介质也具有昼夜节律变化。而下丘脑-垂体-肾上腺轴(hypothalamic-pituitary-adrenal axis, HPA轴)在“同步”靶器官（鼻黏膜）和靶细胞（MC）的昼夜活动节律中起重要作用。为此，本课题后续延伸设计小鼠阻断HPA轴，探讨AR发病中可能的神经内分泌机制。. 联合建立肾上腺切除小鼠模型和AR小鼠模型，采用症状学评分、免疫学检测、鼻黏膜HE染色和甲苯胺蓝染色观察MC和嗜酸性粒细胞（EOS）变化以及流式细胞术检测外周血CD4+T淋巴细胞IL-4和IFN-γ表达率等方法，初步验证双侧肾上腺切除导致HPA轴功能缺陷可能通过调控Th1/Th2免疫平衡影响AR进程，为下一步研究MC脱颗粒的神经内分泌机制做铺垫。