天然免疫识别细菌生物膜在慢性鼻窦炎发病机制中的作用

Chronic rhinosinusitis(CRS) is a common and frequently-occurring disease, however, its pathogenesis has not been fully clarified yet. In recent years, many studies have suggested that bacterial biofilms play a key role in the occurring course of this disease, but the reasons cause the topical nasal inflammation are still in gestation. Our early work have found that the those biofilm+CRS patients have their nasal TLR2 receptor and NF-κB expression elevated as compared with the biofilm-CRS patients.Under the context of the state-of- the- art research at home and abroad, also based on the preliminary work, this research work proposed that , it is the response between bacterial biofilms and the local innate immune system, that breaks the nasal local immune homeostasis, therefore leads to the lasting persistence of chronic nasal inflammation. Due to this proposal, our new experiment will be carried out in the following steps: First, single-species bacterial biofilm and planktonic strains were cultivated in vitro, and then, they were used to stimulate the human nasal epithelial cells. Second, detecting Toll-like receptors (TLR) and downstream inflammatory cytokine expression, and observing of the epithelial cells responding with same species planktonic bacteria and with of the bacterial biofilms, respectively. After this, the RNA interference technology is employed to inhibit the TLR expression of the nasal epithelial cells, to further explore the source role of the innate immune system plays in the interactions between bacterial biofilms and the body. Meanwhile, combined with experimental animal models, to further reveal the interaction of bacterial biofilms and the innate immune system.In conclusion, the above research work has significantly meanings in clarifying the pathogenic role of bacterial biofilms, is important and innovative enough to provide a new approach to explain the pathogenesis of chronic rhinosinusitis.

慢性鼻窦炎为常见多发病，其发病机制尚未明确。近年来许多研究认为细菌生物膜在其发病过程中起了关键作用，但其如何导致鼻腔局部炎症反应尚不清楚。我们发现细菌生物膜阳性的慢性鼻窦炎患者，鼻粘膜TLR2受体和NF-κB表达升高。本课题综合国内外研究和前期工作基础，提出假设：鼻腔致病菌细菌生物膜与局部天然免疫系统相反应，打破鼻腔局部的免疫稳态，导致鼻腔慢性炎症的持续存在。我们拟体外培养的单菌种细菌生物膜及浮游菌株，以分别刺激人鼻粘膜上皮细胞，检测Toll样受体（TLR）及下游炎症因子的表达，观察上皮细胞对同菌种的浮游菌和细菌生物膜的不同反应。并利用RNA干扰技术抑制鼻粘膜上皮细胞TLR的表达，探索天然免疫系统在细菌生物膜与机体相互反应中的源头作用。同时结合实验动物模型，进一步揭示细菌生物膜与天然免疫的相互作用。这对研究细菌生物膜的致病作用有重要意义，为阐明慢性鼻窦炎的发病机制提供了新的思路。

本课题的目的是研究鼻腔致病菌细菌生物膜与局部天然免疫系统相反应，探讨病原微生物如何打破鼻腔局部的免疫稳态,从而导致鼻腔慢性炎症的持续存在。具体目标是：1.体外培养鼻黏膜上皮细胞，并培养单菌种的细菌生物膜；2.观察在单菌种细菌生物膜及浮游菌刺激时，上皮细胞的TLR 受体表达及下游炎症因子的释放；3.对TLR进行RNAi，观察鼻黏膜上皮细胞对单菌种细菌生物膜及浮游菌的反应差异。4.建立基因沉默的动物模型。通过进一步查阅文献结合我们自身实验条件，我们对研究内容进行了调整。鼻腔局部细菌组成的变化有可能会影响细菌的存在方式，因此研究鼻腔局部微生物群的组成，以及成分和形态的相关性有助于深入认识鼻腔微环境，从而进一步了解局部微生物与机体天然免疫系统的相互作用。本研究共纳入39例慢性鼻窦炎患者，9例健康对照组患者，应用微生物群16S-rRNA基因序列测序技术，对分泌物进行鼻腔微生物群的检测。发现慢性鼻窦炎患者的鼻腔微生物群结构与健康人之间有差异，同时结果表明慢性鼻窦炎患者鼻腔内蓝藻门的相对丰度值较健康人高，我们推测蓝藻门可能与慢性鼻窦炎的发病有关，它可能在局部微生态环境的起了重要作用。课题组选取16例慢性鼻窦炎患者、5例对照组患者的钩突黏膜进行电镜扫描、HE染色等观察。结果显示慢性鼻窦炎患者鼻黏膜上皮细胞化生，黏膜下腺体扩张、炎性细胞聚集，骨质增生，可见成骨及破骨细胞。细菌以生物膜的形式粘附在纤毛表面。但目前尚未发现细菌生物膜的检出与哪些细菌相关，需进一步加大例数进行观察。同时本研究采用荧光定量PCR检测实验组和对照组的TLR受体基因，但提取RNA过程失败，分析原因考虑在样本保存、运输过程中出现了RNA降解，结合本课题组有该方面前期研究基础，拟在吸取本次失败经验的基础上调整实验条件继续完成该部分实验。囿于实验条件限制，体外实验目前还在条件摸索中。