药学学报

基于香烟烟雾提取物气管滴注构建一种新的COPD小鼠模型研究

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殷鑫 , 詹红艳 , 刘欣玉 , 李小顺 , 张梓倩 , 李严严 , 林明宝 ^*

药学学报 | 研究论文 2025,60(6): 1746-1754

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药学学报 |研究论文 2025 , 60 (6) : 1746 -1754

基于香烟烟雾提取物气管滴注构建一种新的COPD小鼠模型研究

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殷鑫, 詹红艳, 刘欣玉, 李小顺, 张梓倩, 李严严, 林明宝^*

作者信息

中国医学科学院、北京协和医学院药物研究所, 北京 100050

通讯作者:

林明宝，Tel: 86-10-63165191, E-mail: mingbaolin@imm.cams.cn

作者简介:

作者贡献:

殷鑫负责文献查阅、实验方案设计、实验操作、数据分析作图及论文撰写; 詹红艳协助实验操作及数据分析作图; 刘欣玉和李小顺协助文献查阅; 张梓倩和李严严负责实验方案优化及指导实验; 林明宝指导研究思路、实验方案设计、写作并进行论文修改。

A new mouse model of chronic obstructive pulmonary disease based on intratracheal instillation of cigarette smoke extract

Xin YIN, Hong-yan ZHAN, Xin-yu LIU, Xiao-shun LI, Zi-qian ZHANG, Yan-yan LI, Ming-bao LIN^*

Affiliations

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2025-06-12 doi: 10.16438/j.0513-4870.2024-1287

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摘要

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针对传统的香烟烟雾诱导的慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD) 小鼠模型周期长且稳定性不易保证, 本文基于香烟烟雾提取物(cigarette smoke extract, CSE) 气管滴注, 探讨建立一种CSE诱导的COPD小鼠模型。以C57BL/6J小鼠为模型动物, 除正常对照组外, 各组动物气管滴注CSE, 采用小动物呼吸肺功能检测系统测定小鼠气道反应性及肺功能, 血液体液分析仪测定小鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF) 中白细胞分类计数, 酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA) 测定BALF中炎症因子及中性粒细胞胞外诱捕网(neutrophil extracellular traps, NETs) 水平, 以及苏木精-伊红染色法(hematoxylin-eosin staining, H&E) 检测小鼠肺组织病理学改变。实验经中国医学科学院、北京协和医学院药物研究所实验动物管理与动物福利伦理委员会审批通过(批准号: 00006301)。研究结果表明, 气管滴注CSE后, 模型组小鼠呈现显著的COPD样病理和功能性改变, 表现为呼吸急促、体重增长缓慢; H&E染色见肺组织大量炎性浸润、肺泡壁塌陷及肺泡腔扩张等病理变化; BALF中炎症细胞分类计数(白细胞总数、中性粒细胞数、嗜酸性粒细胞数)、炎症因子水平如肿瘤坏死因子α (tumor necrosis factor-α, TNF-α)、白介素6 (interleukin-6, IL-6) 及NETs水平与正常对照组比较均显著升高(P < 0.01、P < 0.001); 气道反应性指标特殊气道阻力(specific resistance, airway, sRaw) 显著升高(P < 0.05), 肺功能指标呼气峰值流速(peak expiratory flow, PEF)、用力呼气75%肺活量的瞬间流量(forced expiratory flow at 75% of forced vital capacity, FEF₇₅)、动态肺顺应性(dynamic lung compliance, Cdyn)、第100 ms用力呼气量(forced expiratory volume in the first 100 ms, FEV₁₀₀) 及第100 ms用力呼气量占用力肺活量的比值(the ratio of forced expiratory volume in the first 100 ms to forced vital capacity, FEV₁₀₀/FVC) 均显著降低(P < 0.01), 呼吸指数(respiratory index, RI) 显著上升(P < 0.05)。而与模型组相比, 罗氟司特(roflumilast, RFST) 可显著改善模型动物的炎症病理改变和肺功能损伤。综上, 基于CSE气管滴注可快速制备COPD小鼠模型, 在一定程度上复制COPD气道炎症及肺功能下降, 可为COPD病理机制、药物筛选评价研究提供一种新的候选动物模型。

关键词

动物模型 / 慢性阻塞性肺疾病 / 香烟烟雾提取物 / 气道炎症 / 肺功能

Abstract

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The traditional cigarette smoke-induced chronic obstructive pulmonary disease (COPD) mouse model has a long period and its stability is not easily guaranteed. Based on the intratracheal instillation of cigarette smoke extract (CSE), this study aims to establish a CSE-induced COPD mouse model. C57BL/6J mice were used as model animals, except for the normal control group, animals in each group received intratracheal instillation of CSE. The airway reactivity and pulmonary function of the mice were measured using a small animal respiratory pulmonary function testing system, the leukocyte classification count in the bronchoalveolar lavage fluid (BALF) of the mice was determined using a blood and body fluid analyzer, the levels of inflammatory factors and neutrophil extracellular traps (NETs) in BALF were measured using enzyme-linked immunosorbent assay (ELISA), and the histopathological changes in the mouse lung tissue were detected using H&E staining method. The experiments were approved by the animal care and welfare committee of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (approval No. 00006301). The results of the study showed that after intratracheal instillation of CSE, the model group mice exhibited significant COPD-like pathological and functional changes, characterized by tachypnea, slow weight gain. H&E staining showed pathologic changes such as massive inflammatory infiltration of lung tissue, alveolar wall collapse and alveolar lumen dilatation. The leukocyte classification count (total leukocytes, neutrophils, eosinophils), the levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and NETs levels in the BALF were significantly increased compared to the normal control group (P < 0.01, P < 0.001). The index of airway reactivity, specific resistance, airway (sRaw) was significantly increased (P < 0.05), pulmonary function indicators such as peak expiratory flow (PEF), forced expiratory flow at 75% of forced vital capacity (FEF₇₅), dynamic lung compliance (Cdyn), forced expiratory volume in the first 100 ms (FEV₁₀₀), and the ratio of forced expiratory volume in the first 100 ms to forced vital capacity (FEV₁₀₀/FVC) were significantly reduced (P < 0.01), while the respiratory index (RI) was significantly increased (P < 0.05). Compared to the model group, roflumilast can significantly improve the inflammatory pathological changes and pulmonary function damage in model animals. In summary, the intratracheal instillation of CSE can rapidly establish a COPD mouse model, replicating the airway inflammation and decreased pulmonary function of COPD to a certain extent, and provides a new candidate animal model for the study of the pathological mechanism and drug screening evaluation of COPD.

Key words

animal model / chronic obstructive pulmonary disease / cigarette smoke extract / airway inflammation / pulmonary function

引用本文

殷鑫, 詹红艳, 刘欣玉, 李小顺, 张梓倩, 李严严, 林明宝. 基于香烟烟雾提取物气管滴注构建一种新的COPD小鼠模型研究. 药学学报, 2025 , 60 (6) : 1746 -1754 . DOI: 10.16438/j.0513-4870.2024-1287

Xin YIN, Hong-yan ZHAN, Xin-yu LIU, Xiao-shun LI, Zi-qian ZHANG, Yan-yan LI, Ming-bao LIN. A new mouse model of chronic obstructive pulmonary disease based on intratracheal instillation of cigarette smoke extract[J]. Acta Pharmaceutica Sinica, 2025 , 60 (6) : 1746 -1754 . DOI: 10.16438/j.0513-4870.2024-1287

正文

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慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD) 是一种由气道炎症和/或肺泡结构异常导致持续的、进行性的气流阻塞而产生的常见的慢性呼吸系统疾病^[1], 已成为全球第三大致死疾病^[2]。在中国, COPD疾病负担呈现患病率高、死亡率高、经济负担高的“三高”特征, 患病人数约占全球总数的四分之一。而且, 随着人口老龄化、吸烟持续高发和环境空气污染的影响, 我国COPD的疾病负担预计将继续大幅增加, 给社会和经济带来沉重负担^[3]。然而, COPD药物治疗当前仍面临巨大挑战。虽然当前临床治疗主要手段吸入性糖皮质激素和长效β2受体激动剂、双支扩与三联疗法有效缓解了COPD患者的疾病症状, 但仍存在许多不足, 如感染、耐药、黏膜出血及内分泌代谢异常等不良反应。因此, 发现新的COPD治疗药物十分紧迫。

但是, 当前对于COPD研究及药物评价的疾病动物模型仍十分有限, 现有的COPD动物模型诱导因素包括香烟烟雾(cigarette smoke, CS)、弹性蛋白酶、脂多糖、细菌和空气颗粒物等^[4]。由于吸烟被认为是COPD的最强刺激因素^[5], CS暴露模型作为单因素诱导的典型代表应用最为广泛, 该模型较好地复制了COPD气道慢性炎症和小气道功能受损的病理特征, 但其存在造模周期长且稳定性不易保证等不足。单一的动物模型无法模拟人类COPD的复杂疾病特征, 多种疾病模型的联合评价可有效帮助研究人员加深对COPD病理机制的探索以及相关治疗药物的筛选与评价研究。而且, 实验动物模型作为COPD病理机制、药物发现研究的重要载体, 其病理特征和发生发展规律的准确性和可靠性显得尤为重要。香烟烟雾提取物(cigarette smoke extract, CSE) 常作为刺激剂用于COPD相关体外细胞模型的建立^[6], 有研究报道其通过鼻腔滴注或腹腔注射建立动物模型用于COPD研究^[7-10], 但是鼻腔滴注主要影响上呼吸道且暴露不均匀, 腹腔注射诱导的病理特征与COPD肺部的疾病规律存在较大的差异。因此, 本研究采用气管滴注CSE方法, 制备COPD小鼠模型, 并从气道炎症、肺组织病理、气道反应性及肺功能等指标评价模型的可行性, 以期为COPD病理机制及药物发现研究提供一种周期短、稳定可靠, 且更符合COPD肺部疾病特征的动物模型, 为现有模型提供一种有效补充。

材料与方法

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主要试剂与仪器 “大前门”牌香烟(焦油量9 mg, 烟气烟碱量0.6 mg, 烟气一氧化碳量12 mg) 购自上海烟草集团有限责任公司; 罗氟司特(roflumilast, RFST) 购自上海源叶生物科技有限公司(货号S43303); 小鼠肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)、白介素-6 (interleukin-6, IL-6) 酶联免疫吸附检测试剂盒(enzyme-linked immunosorbent assay, ELISA) 均购自美国Biolegend公司(货号430904, 431304); Quant-iT^TM PicoGreen^TM dsDNA定量试剂盒购自美国Invitrogen公司(货号P7589); 氯化乙酰甲基胆碱(acetyl-beta-methylcholine chloride, Mch) 购自德国Sigma公司(货号A2251)。

Sysmex XN1000全自动血液体液分析仪, 日本希森美康公司; Buxco小动物呼吸肺功能检测系统, 美国DSI公司; BX 43生物显微镜, 日本OLYMPUS公司; 包埋机、切片机、染色机, 德国Leica公司; 3K15高速冷冻离心机, 德国Sigma公司; GL-802A型微型台式真空泵, 海门市其林贝尔仪器制造有限公司。

实验动物 雄性C57BL/6J小鼠, SPF级, 体重20~22 g, 购买于北京华阜康生物科技股份有限公司, 动物许可证编号: SCXK (京) 2024-0003。动物饲养于中国医学科学院、北京协和医学院药物研究所动物实验中心, 饲养条件: 室温23 ± 2 ℃, 湿度50% ± 10%, 通风10次/小时全新风, 12 h/12 h光暗循环, 自由进食进水。本研究由中国医学科学院、北京协和医学院药物研究所实验动物管理与动物福利伦理委员会审批通过(动物伦理批准号: 00006301)。

CSE的制备 采用负压抽吸的方式制备CSE生理盐水溶液^{[11, 12]}。于包氏管中装入生理盐水; 顶端连接香烟, 侧端连接真空泵后, 点燃香烟, 控制真空泵匀速、缓慢抽吸香烟烟雾至生理盐水中, 直至香烟燃尽; 每燃尽1支香烟后, 轻轻晃动包氏管使烟雾充分溶于生理盐水, 制得CSE终浓度为每毫升含1支香烟; 液体移入离心管以5 000 r·min^-1离心10 min, 取上清(即为CSE), -80 ℃冻存备用。

实验分组和COPD模型制备 如图1所示, 30只雄性C57BL/6J小鼠随机分为3组: 对照组、模型组、罗氟司特组, 每组10只。除对照组给予等量生理盐水外, 模型组、罗氟司特组小鼠分别于第1、5、9天麻醉后, 气管滴注50 μL CSE制备疾病模型。同时, 罗氟司特组于第3~9天, 5 mg·kg^-1腹腔注射(intraperitoneal injection, ip) 给药, 每天1次, 连续7天, 对照组和模型组给予等剂量的生理盐水。动物末次给药24 h后(即第10天), 进行有关指标的检测。

小鼠一般情况观察及体重监测 每天观察小鼠饮食进水、精神状态、呼吸频率等行为, 每两天测定小鼠体重1次, 监测各组小鼠体重的动态变化。

小鼠气道反应性测定 清醒小鼠置于无创特殊气道阻力检测系统(Buxco-NAM) 中, 使其适应5 min后, 生理盐水(0.9% NaCl) 或递增浓度的氯化乙酰甲基胆碱(6.25、12.5、25 mg·mL^-1) 雾化并送入腔室1 min, 每次给药后记录2 min内的特殊气道阻力(specific resistance, airway, sRaw) 测量值并取其平均值, 用于比较各组小鼠气道反应性的差异。

小鼠肺功能测定 小鼠麻醉后, 分离颈部皮肤与肌肉, 行气管插管; 插管另一侧连接肺功能检测系统(Buxco-PFT), 检测呼气峰值流速(peak expiratory flow, PEF)、用力呼气75%肺活量的瞬间流量(forced expiratory flow at 75% of forced vital capacity, FEF₇₅)、动态肺顺应性(dynamic lung compliance, Cdyn)、第100 ms用力呼气量(forced expiratory volume in the first 100 ms, FEV₁₀₀)、第100 ms用力呼气量占用力肺活量的比值(the ratio of forced expiratory volume in the first 100 ms to forced vital capacity, FEV₁₀₀/FVC, 其中FVC经体重校准), 以及呼吸指数(respiratory index, RI)。

支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中白细胞总数及其分类计数 采用预冷PBS溶液(分3次, 共2 mL) 收集BALF, 以1 500 r·min^-1、4 ℃离心5 min, 取上清待测炎症因子, 细胞沉淀以200 μL添加有5 mg·mL^-1 EDTA·2K的PBS溶液重悬, 通过Sysmex XN1000全自动血液体液分析仪测定BALF中白细胞(leukocyte, white blood cell, WBC) 总数及其中性粒细胞(neutrophil, Neu)、淋巴细胞(lymphocyte, Lym)、单核细胞(monocyte, Mon)、嗜酸性粒细胞(eosinophil, Eos)、嗜碱性粒细胞(basophil, Bas) 数量。

BALF炎症因子测定 取BALF上清, 采用ELISA法, 参照试剂盒说明检测BALF中TNF-α、IL-6的含量。

BALF中性粒细胞胞外诱捕网(neutrophil extracellular traps, NETs)含量测定 采用Quant-iT^TM PicoGreen^TM dsDNA定量试剂盒, 测定BALF中NETs的主要成分游离DNA (cell-free DNA, cfDNA) 含量^[13]。

肺组织病理学检测 取小鼠肺组织, 于4%甲醛中固定后, 脱水, 石蜡包埋, 切片后进行苏木精-伊红染色(hematoxylin-eosin staining, H&E), 做病理组织学检查; 并对其支气管-血管周围炎症进行评分(0: 未见炎症细胞浸润; 1: 偶见静脉、支气管旁袖口状炎症细胞浸润; 2: 大多数静脉、支气管旁炎症细胞浸润明显, 炎症细胞1~5层; 3: 大多数静脉、支气管旁大量炎症细胞浸润, 炎症细胞大于5层), 每张切片选择5处进行评分, 计算其平均值。

统计学分析 采用SPSS Statistics 26.0统计软件进行数据统计分析, 数据以平均值±标准差(x ± s) 表示; 组间数据比较采用单因素方差分析LSD检验(方差齐) 或Tamhane's T2检验(方差不齐), 以P < 0.05判定具有统计学差异。

结果

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1　模型小鼠一般情况观察

正常对照组小鼠实验期间活动饮食均正常, 毛发光泽, 精神可, 呼吸频率正常, 无喘息等情况; 模型组小鼠饮食减少, 呼吸急促伴喘鸣, 精神倦怠; 罗氟司特组可改善上述症状。

2　模型小鼠体重的变化

如图2结果所示, 与正常对照组相比较, 模型组小鼠体重增长缓慢, 第8天起, 模型组体重与对照组相比体重显著下降(P < 0.05、P < 0.01), 与COPD患者及传统COPD小鼠模型的体重变化趋势一致。而且, 阳性药罗氟司特干预后, 给药组较模型组小鼠体重下降显著(P < 0.05、P < 0.01、P < 0.001), 这也与临床报道的罗氟司特用于COPD患者治疗, 存在体重减轻的不良反应相一致^[14]。

以上结果表明: 气管滴注CSE可引发小鼠产生COPD呼吸急促、喘息的典型症状, 以及食欲不振、体重下降的伴随症状。

3　模型小鼠BALF中白细胞分类计数

如图3结果所示, 与正常对照组相比较, 模型组小鼠BALF中白细胞总数、中性粒细胞、嗜酸性粒细胞计数显著升高(P < 0.01); 而且, 中性粒细胞占比升高显著(P < 0.001), 与COPD气道炎症以中性粒细胞为主要炎症浸润细胞的病理特征相符; 此外, 与模型组相比较, 罗氟司特组可显著降低模型小鼠白细胞总数、中性粒细胞、单核细胞、嗜酸性粒细胞、嗜碱性粒细胞计数以及中性粒细胞占比(P < 0.01)。

4　模型小鼠BALF中炎症因子及NETs水平

如图4结果所示, 与正常对照组相比较, 模型组小鼠BALF中炎症因子TNF-α、IL-6生成水平, 以及NETs释放水平均显著升高(P < 0.01、P < 0.001), 模型小鼠肺泡炎症反应特征与传统COPD模型特征相符; 与模型组相比较, 罗氟司特组可显著降低模型小鼠TNF-α、IL-6以及NETs水平(P < 0.01、P < 0.001), 显示其治疗效果。

以上结果表明: 气管滴注CSE可引发小鼠气道炎症, 中性粒细胞、TNF-α及IL-6是主要的炎症细胞和细胞因子, 与COPD气道炎症特征相符, 说明模型构建成功; 同时, 该模型还观察到NETs水平的升高。

5　模型小鼠肺组织病理学检查结果

如图5结果所示, 光镜下, 正常对照组小鼠肺组织结构形态学正常, 肺泡清晰, 未见明显炎症细胞浸润; 模型组小鼠肺组织部分肺泡壁塌陷, 肺泡不规则扩大并相互融合, 细支气管、小静脉周围见大量炎症细胞浸润, 气道上皮细胞纤毛发生粘连、坏死以及脱落等改变, 炎症病理评分与正常对照组相比较显著升高(P < 0.001); 罗氟司特组可显著改善模型小鼠肺组织的病理损伤、显著降低其炎症病理评分(P < 0.05或P < 0.001)。

该结果表明: 气管滴注CSE可引发小鼠肺组织炎症细胞浸润、肺泡结构破坏等COPD肺组织损伤的病理学形态改变, 进一步说明模型构建成功。

6　模型小鼠气道反应性(支气管激发试验)

如图6结果所示, 随着氯化乙酰甲基胆碱浓度的成倍递增, 模型组与正常对照组小鼠的sRaw均逐渐增加, 当浓度达到25 mg·mL^-1时, 模型组小鼠sRaw显著高于正常对照组(P < 0.01), 提示模型小鼠气道反应性增高; 当浓度达到12.5 mg·mL^-1以上时, 罗氟司特可显著降低模型小鼠sRaw (P < 0.05、P < 0.001)。

该结果表明: 气管滴注CSE可使小鼠对氯化乙酰甲基胆碱刺激产生过强的支气管收缩反应, 引发气道反应性增高, 进一步说明模型构建成功。

7　模型小鼠肺功能测定结果

如图7结果所示, 与正常对照组相比较, 模型组小鼠肺功能流量-容积曲线(flow-volume loop, F-V loop) 减弱、曲线下面积显著减小; 如图8结果所示, 模型小鼠PEF、FEF₇₅、Cdyn、FEV₁₀₀以及FEV₁₀₀/FVC均显著降低(P < 0.01、P < 0.001), 而RI显著上升(P < 0.05), 提示模型小鼠肺功能显著下降, 与COPD患者临床肺功能特征相符; 此外, 罗氟司特可增强模型小鼠肺功能F-V loop, 显著提升PEF、FEF₇₅、Cdyn、FEV₁₀₀等肺功能指标(P < 0.05), 显著降低RI (P < 0.05)。

该结果表明: 气管滴注CSE可引发小鼠肺通气功能障碍、肺弹性降低等COPD样肺功能损伤, 进一步说明模型构建成功。

讨论

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疾病动物模型在人类疾病病理机制及其治疗药物发现研究中具有十分重要的作用, 理想的动物模型应尽可能复制人类疾病的特点和发生发展过程, 此外还必须具备可重复性高、同源性高、动物成活率高等条件。诸如吸烟、遗传、变应原、污染物、病原体等的环境暴露是COPD的主要风险因素, 尤其是吸烟已成为COPD最强的刺激因素^[15], CS以多种方式损伤人体, 更容易引起肺功能异常, 其有害成分在肺部沉积, 引发气道炎症, 导致肺组织结构改变和功能异常、气道阻力升高, 促进COPD的发展^[16]。

长期CS暴露是最常应用的COPD疾病动物模型单因素造模方法, 真实地模拟了人类患病环境, 且符合人类吸烟过程, 但在停止刺激后疾病不同于人类所呈现的进行性发展特点且部分可逆^[17], 无法模拟临床COPD的复杂性。此外, 由于其造模时间通常持续6个月左右^[18], 增加了实验的复杂性和成本, 由于CS的暴露量和暴露均匀性难以精确控制且缺乏统一标准, 限制了该模型的应用。另有研究报道, CSE鼻腔滴注和腹腔注射方式与CS暴露的建模效果相近, 鼻腔滴注可以模拟烟雾等有害物质通过鼻腔进入肺部的自然过程, 操作相对简便, 刺激物可通过鼻腔分布到上呼吸道, 但其在鼻腔的吸收受到多种因素影响, 且到达下呼吸道的量不足, 加之可能引起鼻部疾病, 导致模型一致性较差, 影响COPD模型构建效果。虽然CSE含有香烟烟雾的所有成分, 但是其腹腔注射所涉及的病理生理机制仍不清楚, 且与CS肺部暴露模型条件存在明显差异, 究竟是靶向性损伤肺组织还是诱导全身性炎症细胞通过受损内皮向肺组织浸润有待明确^[19], 影响该方法制备的模型对COPD疾病特征的复制。

对此, 为克服CS肺部暴露制备COPD模型周期长、CSE鼻腔滴注一致性差且具有不良反应、CSE腹腔注射制备模型病理生理不清的不足, 本研究采用短期多次气管滴注CSE方法, 建立了一种CSE肺部直接靶向性损伤的新型小鼠COPD模型, 并全面评价了该模型对COPD疾病特征的复制性及其用于疾病研究的可行性。总的来说, 本研究所构建的COPD动物模型, 与传统的长期CS暴露相比, 具有短期内快速重现COPD大部分病理特征的优势; 与CSE鼻腔滴注相比, 具有一致性和重复性好、能够重现COPD小气道阻塞病理特征的优势; 与CSE腹腔注射相比, 模拟了烟雾对肺部的直接影响, 减少了全身不良反应, 具有更贴近COPD病理机制的优势。

慢性咳嗽、呼吸急促和喘息是COPD患者主要的临床症状, 体重减轻也是重要的临床表现之一, 与频繁的病情恶化和意外再入院的风险增加有关^[20]。能量失衡引起的COPD患者体重减轻可能是呼吸功增加、慢性炎症、急性加重和厌食症导致的能量摄入减少所致^{[21, 22]}。本研究结果显示, CSE气管滴注模型组相较于正常对照组小鼠呼吸急促伴喘鸣、体重增长缓慢, 这与临床COPD患者表现一致。

气道炎症贯穿COPD的整个病程, 中性粒细胞处于COPD气道炎症反应的中心, 大量研究证实COPD患者体内中性粒细胞数量增加或功能激活, 表现为痰液和BALF中的中性粒细胞数量增加^{[23, 24]}、中性粒细胞肺部迁移和黏附增加^[25]。本研究结果显示, CSE气管滴注模型组小鼠BALF中性粒细胞占比高达90%, 这与吸烟引起COPD的中性粒细胞炎症相符。而且, 研究报道中性粒细胞NETs存在于COPD稳定期及急性加重期患者的痰中, 且与气流受限严重程度相关^[26], 这可能与香烟烟雾和烟草主要成分尼古丁可以引起中性粒细胞释放NETs有关^[27]。本研究制备的模型组小鼠BALF中NETs主要成分cfDNA含量显著升高, 进一步提示该模型与吸烟引起的COPD特征相符。此外, 吸烟引起的中性粒细胞炎症及巨噬细胞活化释放了大量炎症因子, 本研究模型中检测到异常升高的TNF-α和IL-6, 可以刺激中性粒细胞释放NETs, 而NETs及其成分与激活先天免疫应答反应有关^[28], 如此循环互作放大COPD的炎症反应。进一步采用最直观的肺组织病理评价指标^[29], 评价本研究模型小鼠肺组织的病理改变。研究结果显示, 模型小鼠肺组织支气管、血管周围区域表现出广泛的炎症细胞浸润、肺泡壁塌陷、肺泡扩大融合形成肺大泡且伴有气道上皮细胞坏死及脱落, 而且炎症病理评分显著升高, 这些病理改变均与人类COPD的病理特征相似。综上可见, 本研究采用CSE气管滴注制备的小鼠模型可有效复制COPD气道炎症病理生理特征。

气道高反应性(airway hyperresponsiveness, AHR) 是哮喘的主要特征, 在COPD患者中也存在^[18], 且与预后变差、死亡风险增加相关^[30]。此外, 不仅在哮喘动物模型中, 在多种COPD动物模型中也观察到对不同刺激的AHR^{[31, 32]}。在本研究中, CSE气管滴注模型组小鼠对乙酰甲胆碱的敏感性增加并导致AHR, 这可能是气道炎症及肺实质结构改变引起的。

肺功能是临床上诊断COPD的金标准, 进行性肺功能下降是COPD的标志, 同时也是评价动物模型的重要指标。小气道是COPD的主要气流阻塞部位^[33], 导致第一秒用力呼气量(forced expiratory volume in the first second, FEV₁) 和FEV₁/FVC比值的降低, 并随着时间推移而发展; 肺气肿时, 肺泡间隔丧失^[34], 肺充气不均, Cdyn减低; COPD患者F-V loop相较于正常人有所“塌陷”^[35], 在本研究中, CSE气管滴注模型组小鼠观察到同样的肺功能改变现象: F-V loop“塌陷”, PEF、FEF₇₅、FEV₁₀₀、FEV₁₀₀/FVC及Cdyn均显著下降, 提示小气道功能障碍、气道阻塞; RI升高, 提示气道阻塞导致肺部通气功能和换气功能存在障碍, 与既往COPD疾病临床特征的研究结果相符^{[36, 37]}。AHR及肺功能检测结果进一步证明了本研究所制备的动物模型对COPD典型小气道阻塞疾病特征的有效复制。

综上所述, 本研究基于CSE气管滴注方法建立的COPD小鼠模型, 可在一定程度上复制COPD的气道炎症和以小气道阻塞为代表特征的肺功能下降。而且, COPD治疗药物罗氟司特在该模型中表现出显著的治疗效果。因此, 可以认为气管滴注CSE能够成功构建COPD小鼠模型, 该模型的优点在于耗时短、易于操作、成本低而且可复制COPD的大部分病理特征, 该模型可为COPD病理机制及药物治疗筛选评价研究提供一种候选的疾病动物模型工具。

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2025年第60卷第6期

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doi: 10.16438/j.0513-4870.2024-1287

接收时间：2024-12-25
首发时间：2026-09-20
出版时间：2025-06-12

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收稿日期：2024-12-25
修回日期：2025-02-18

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中国医学科学院、北京协和医学院药物研究所, 北京 100050

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林明宝，Tel: 86-10-63165191, E-mail: mingbaolin@imm.cams.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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