肺泡巨噬细胞功能活性在早期慢阻肺戒烟前后肺泡及小气道结构改变的作用机制研究

Heavy smoking has been a risk factor for developing COPD, it could also cause alveolar macrophage (AM) dysfunction and imbalance, which play an important role in the mechanism of airway inflammation and remodeling in COPD. Smoking cessation may have a reversible effect on AM function, airway inflammation, small airway remodeling and alveolar disorders in early stage COPD. As endobronchial optical coherence tomography (OCT) provides a high-resolution measurement for microstrcuture of small-airway, we have previously demonstrated the small-airway disorders in early stage COPD. Confocal laser microendoscopy (CLE) can be performed in real-time and obtained structure imaging of airway epithelium, alveoli, fibers and cellular in vivo. This study aims to elucidate the small-airway and alveolar structure in early stage COPD by using OCT and CLE. Along with OCT and CLE measurements, counts of AM in bronchoalveolar lavage fluid, AM phenotype (M1/M2), as well as cytokines expression (MMP-9 and IL-10) would be analyzed to explore the correlation between AM function and structural characteristics of small airway and alveoli in early stage COPD. The role of AM in airway remodeling in population at early stage COPD would be verified based on the comparison of the changes between functional activity of AM and structure of small airway and alveoli before and 1 year after smoking cessation.

吸烟是慢阻肺的高危因素，导致肺泡巨噬细胞（AM）功能活性失衡是慢阻肺发生气道炎症和结构重塑的重要机制。戒烟可能对早期慢阻肺AM功能、气道炎症、小气道重塑和肺泡结构改变具有可逆改善作用。前期研究中我们利用光学相干断层扫描（OCT）显示小气道结构，发现早期慢阻肺已出现明显小气道病变；并通过共聚焦激光显微内镜（CLE）观察活体内气道上皮、肺泡、纤维及细胞结构。本研究拟（1）通过气道OCT及肺泡CLE明确早期慢阻肺小气道及肺泡结构特征；（2）对早期慢阻肺患者肺泡灌洗液巨噬细胞计数、细胞表型（M1/M2型）及吞噬和分泌（MMP-9、IL-10）功能检测，结合OCT和CLE显像，明确AM功能活性与小气道和肺泡结构的相关性；（3）明确戒烟1年前后小气道和肺泡结构及AM功能活性变化，及其在早期慢阻肺小气道重塑和肺泡结构病变中的作用机制，并验证戒烟通过影响AM功能活性改善早期慢阻肺小气道重塑。

重度吸烟是慢阻肺发生发展的重要危险因素，筛选识别慢阻肺小气道病变的生物学和光学成像指标有助于疾病的早期诊断。本研究基于传统的肺泡灌洗液（BALF）中肺泡巨噬细胞提纯离心法和贴壁法，综合运用裂痰、物理过滤、密度梯度离心的方法提纯肺泡灌洗液中的细胞，大大减少细胞碎片或其他杂质的干扰，保留细胞种类的完整性、减少细胞量的损失，为后续细胞学实验建立方法学基础。通过流式细胞学分析结果显示，吸烟患者的肺泡巨噬细胞百分率显著高于非吸烟人群（分别为95.8% 和74.2%，P<0.01)，其中CD206+的M2型肺泡巨噬细胞在小气道功能障碍患者和慢阻肺患者中均明显升高，且其水平与肺功能中FEV1/FVC%预计值呈显著负相关(r=-0.875, P<0.01)；，相对于正常非吸烟人群，慢阻肺患者的CD4+、CD8+T细胞以及CD4/CD8比值均出现显著增加，小气道功能障碍患者和慢阻肺患者肺泡灌洗液中炎症因子IL-1β均明显高于正常非吸烟者。此外，本研究基于支气管分支追踪方法，通过HRCT图像和超细支气管镜构建支气管镜导航的分层时钟刻度手绘图方法，有助于气道内光学相干断层成像（OCT）在靶肺段的精准定位与测量；而通过大数据处理和深度学习训练，构建OCT图像智能自动分析系统，显著提高气道内OCT测量的效率与准确性。以此为基础，利用OCT测量非吸烟者、小气道障碍吸烟者与慢阻肺患者的小气道结构病变，发现慢阻肺小气道表现为显著的结构重塑，表现为管腔狭窄与气道管壁增厚，肺泡融合与破坏。继而，通过OCT实时成像，可评估支气管舒张剂对慢性气道疾病的作用靶部位（主要为中小气道），从而更好地评估药物作用部位及效能。通过热消融气道管壁后，病理及免疫组化结果显示气道重塑较治疗前改善，气道神经纤维与神经因子分布均减少，结合气道内OCT成像的动态评估，进一步揭示慢性气道疾病气道重塑及其治疗的相关作用机制。