【作者】 唐勇军；

【导师】 胡成平； 罗百灵； 苏晓丽；

【作者基本信息】 中南大学 ， 内科学， 2006， 博士

【摘要】 吸烟是人类慢性阻塞性肺疾病（COPD）的主要致病因素，香烟中有害成份首先攻击气道上皮，气道上皮的损伤及炎症是COPD的重要病理特征，气道上皮的功能缺陷与气道疾病的发生发展有密切关系，目前研究证实COPD等气流受限性疾病发病过程中，气道上皮损伤时，炎性细胞活化并浸润于气道，使气道壁感觉神经末梢暴露、气道神经及神经肽的表型和含量均发生变化，众多神经肽如P物质（SP）、降钙素基因相关肽（CGRP）等大量释放，它们通过多种形式影响气道组织并可诱发神经源性炎症，表现为气道腺体分泌增加、血管扩张和血管通透性增加、气道平滑肌收缩、气道炎症细胞活化等。采用单一介质拮抗剂所起治疗作用往往有限，因此研究调节神经肽释放的化学物质可能是阻断慢性炎症发生、发展的重要途径。近年来，神经生长因子（NGF）由于具有高效的调控神经肽作用而受到国内外学者的关注。在多种炎症性疾病中NGF表达增加，NGF由外周靶细胞（包括免疫炎症细胞和组织结构细胞如气道上皮细胞、血管平滑肌细胞、气道平滑肌细胞、成纤维细胞等）合成并释放，并可被神经轴突末梢摄取，逆行运输至背根节，使背根节感觉神经元的敏感性增高，使合成并释放SP、CGRP等增加。我们的前期研究结果显示大鼠腹腔注射NGF后，肺组织中SP的受体即神经激肽受体1（NK-1R）表达上调，提示NGF可调节神经肽释放，应用抗NGF抗体可减轻RSV感染引起的肺组织神经源性炎症，并使肺组织NK-1R表达下调。综上所述，我们提出假说：COPD存在NGF表达增高；NGF通过上调SP等神经肽调控COPD气道神经源性炎症。由此，本实验测定COPD患者NGF水平，并在已建立的COPD模型中检测NGF及SP的变化，进一步用抗NGF干预以观测COPD大鼠下呼吸道及背根节SP水平的改变，旨在探讨NGF在实验性COPD大鼠中作用及初步机制，以寻求人类COPD治疗的新靶点。为证明上述假设我们完成了以下研究内容。第一部分：COPD患者血清NGF变化及与肺功能关系研究本部分对COPD患者体内NGF的变化进行了初步研究，以期进一步探讨和揭示人类COPD发病机制。我们以ELISA法测定COPD急性加重期患者（31例）、稳定期患者（30例）及正常对照组24例血清NGF水平，并检测同期FEV₁／FVC、FEV₁％pre等肺功能指标。结果发现：COPD急性加重期、稳定期患者，正常对照者血清NGF蛋白水平分别为30.3±11.7ng／L，17.2±8.5ng／L，7.29±3.4ng／L。与正常对照者相比，COPD急性加重期、稳定期患者NGF蛋白水平显著升高（均为P＜0.05），且COPD急性加重期患者明显高于稳定期患者（P＜0.05）。无论是COPD急性加重期还是稳定期患者血清NGF蛋白水平与FEV₁％pre、FEV₁／FVC％均呈负相关，在急性加重期分别为rs=-0.73，rs=-0.72（均P＜0.01），在稳定期分别为rs=-0.65，rs=-0.68（均P＜0.05）。研究结果提示NGF可能直接参与或调控COPD炎症，在患者急性加重期与稳定期的整个病程中发挥作用。第二部分：COPD模型的建立首先采用烟熏加气管内注入猪胰弹性蛋白酶的方法建立大鼠慢性阻塞性肺疾病的动物模型。并将Wistar大鼠随机分为模型组、正常对照组，每组15只。并从肺功能测定、HE染色观察肺组织病理变化及BALF炎症细胞计数等多方面来证实模型的成功性。结果发现：与正常对照组比较：（1）肺功能：COPD模型组大鼠PEF（23.61±2.67 ml／s vs 30.96±2.88 ml／s）、FEV_0.3（3.49±0.42ml vs 4.86±0.36 ml）和FEV_0.3／FVC(（62.25±4.84）％vs （86.68±6.12）％]显著下降（均P＜0.01）；（2）病理改变：平均内衬间隔（136.92±16.71μm vs 36.70±4.48μm）明显增高，平均肺泡数（11.07±0.26个／μm² vs 24.40±2.83个／μm²）明显降低（均P＜0.01）；（3）BALF炎症细胞：细胞总数（10⁴／ml）（23.93±3.52 VS 15.27±2.40）、中性粒细胞计数（％）均明显增高（7.56±1.47 VS 1.80±0.68）（均P＜0.01）。研究证实烟熏加气管内滴入猪胰弹性蛋白酶的方法可建立稳定的大鼠慢性阻塞性肺疾病的动物模型，模型组大鼠有气道炎症和肺气肿改变，小气道阻力增加，有阻塞性通气功能障碍。第三部分：NGF在COPD大鼠中的作用及其机制初探。为探讨NGF在COPD大鼠中的作用及机制，同上方法建立COPD模型和分组：正常对照组、COPD组及抗NGF抗体干预组，每组15只。模型建立后测定肺功能、BALF炎症细胞计数并取肺组织采用HE染色观察肺组织病理变化，同时采用ELISA法测定COPD大鼠BALF中NGF水平，免疫组化法观测三组肺组织、背根神经节NGF、SP及肺组织NK-1R变化，原位杂交比较三组背根神经节SP水平，RT-PCR半定量法测定COPD组及正常对照组肺组织、背根神经节NGFmRNA表达，Western-blot法检测三组NGF蛋白水平变化。结果发现：（1）COPD模型组与正常对照组比较：；BALF中NGF蛋白水平（ng／L）显著升高（187.18±64.76 vs 75.13±20.44，P＜0.001）；肺组织及背根神经节NGF蛋白表达无论是免疫组化（平均灰度值104.23±7.74 vs 153.50±8.02，108.95±7.02 vs 171.03±8.14，灰度值越高表明其在细胞中表达的量越低），还是Western blot（400.4±51.6ng／L vs 198.5±46.2ng／L，318.8±52.6ng／L vs144.2±32.7ng／L）表达均明显增高（均P＜0.01）；NGF mRNA在肺组织显著增高（82.07±7.89 vs 51.80±8.34）（P＜0.01），在背根节无显著升高（56.67±6.28 vs 51.87±7.01）（P＞0.05）；NK-1R在肺组织表达显著升高（平均灰度值101.33±6.31 vs 157.67±8.15）（P＜0.01）；SP在背根节表达显著增高（平均灰度值103.40±7.08 vs167.06±7.83）（P＜0.05）；SPmRNA在肺组织无显著升高（平均灰度值165.53±7.30 vs 168.07±7.05）（P＞0.05），在背根节表达显著增高（平均灰度值103.58±9.63 vs 173.37±12.83）（P＜0.01）。（2）抗NGF组与COPD组相比较：肺组织及背根神经节NGF蛋白表达无论是免疫组化（平均灰度值130.78±6.35 vs 104.23±7.74，135.68±7.13 vs108.95±7.02），还是Western blot（297.9±43.3ng／L vs 400.4±51.6ng／L，222.3±46.6ng／L vs 318.8±52.6ng／L）表达均明显降低（均P＜0.01）；NK-1R在肺组织显著降低（平均灰度值138.20±7.99vs 101.33±6.31）（P＜0.01）；SP在背根节表达显著降低（平均灰度值138.00±9.32 vs 103.40±7.08）（P＜0.01）；SPmRNA在背根节显著降低（平均灰度值143.21±11.30 vs 103.58±9.63）（P＜0.01）；（3）COPD组大鼠肺组织N6FmRNA与背根节SPmRNA灰度值表达呈负相关（rs=-0.81，P＜0.001）。（4）抗NGF组与COPD组比较，BALF细胞总数（17.13±3.94 vs 23.93±3.52）、中性粒细胞计数明显下降（4.65±0.80 vs 7.56±1.47）（P＜0.01）；肺功能PEF（27.88±2.98 vs 23.61±2.67）FEV_0.3（3.93±0.25 vs 3.49±0.42）与FEV_0.3／FVC（71.48±5.38 vs 62.25±4.84）显著增高（均P＜0.05）；形态学上，平均内衬间隔明显下降（101.88±19.67 vs 136.92±16.71，P＜0.01）。研究结果；发现NGF参与大鼠COPD形成，首次证实NGF干预能有效抑制COPD大鼠气道神经源性炎症，改善COPD病理变化及气流阻塞，其机制可能与下调气道感觉神经元合成和分泌SP表达有关。上述三个部分研究提示NGF通过调节气道感觉神经元合成和分泌SP表达参与COPD神经源性炎症，在COPD气流阻塞中发挥作用；同时抗NGF干预可下调SPmRNA表达和蛋白分泌水平，减轻COPD气道炎症。因此我们可以这样认为：NGF作为气道神经肽SP的上游调节因子在COPD气道神经源性炎症中具有重要调控作用。更多还原

【Abstract】 Tobacco smoking is the main risk factor for COPD （chronicobstructive pulmonary disease）,smoke directly attack and damage theairway epithelium firstly, the damaged epithelium lesion lead to theexpose of the sensory nerve ending, the release of neuropeptides fromairway nerves leading to inflammatory effects. the abnomalinflammation include neurogenic inflammation developed then. Themolecular mechanisms underlying neurogenic inflammation areorchestrated by a large number of neuropeptides including tachykininssuch as substance P（SP） and neurokinin A, or calcitonin gene-relatedpeptide and endothelin-1,resulting in imflammation reaction, such asmucus excretion, hyperemia,increase in vasopermeability, edema,contraction of airway smooth muscle and immunocyte activation.furthermore, only a limited therapeutical effect be achieved by singleneuropeptide antagon.It is urgent and important to study the upstreamregulating factor to block up the release of the neuropeptide and theoccurrence and exacerbation of the inflammatory state.Recently,NGF（nerve growth factor NGF）,a upstream cytokinepossessing high potency for regulating neuropeptide,is a study focus formany domestic and overseas scholar.many inflammatory diseases presenthigh levels of NGF.after NGF is synthetized and released by target cellshch as inflammatory cell and structure cell（including airway epithelim,vascular smooth muscle cell, airway smooth muscle cell, fibroblastetc.）,NGF can be uptaked by nerve axon periphery and delivered toposterior root ganglion.then the sensitiveness of the sensory neuron inposterior root ganglion enhance,neuropetides such as SP and CGRP （calcitonin gene-related peptide） are synthetized and released withgreat quantity.we find Exogenous NGF upregulated NK-1R（neurokinin-1receptor） expression in the rat lungs,anti-NGF antibody inhibited NK-1Rupregul- ation and neurogenic inflammation in RSV-infected rat lungs inprophase study, indicating that NGF mediates the releases of neuropeptdeand is responsible for the exaggerated neurogenic inflammation in airway.Hence, our objective was to study the interaction between NGF andneuropeptide in COPD. we can hypothesize that nerve growth factor iselevated in COPD,NGF can regulate the neurogenic inflammation in theCOPD through neuropeptides up-regulation. To determine the thought,we checked the quantity of NGF in the serum in COPD patients firstly,then a COPD model and anti-NGF antibody were utilized to research itsmechanism, we completed the studies as follow.Study contents1. NGF changes and the correlation with lung function inCOPD patientsthree groups, including 31 cases in acute exacerbation stage,30 casesin stable stage and normal control group were checked by lung functionand the quantity of NGF in the serum were detected by enzyme-linkedimmunosorbent assay（ELISA）.Results showed: As compared with the control group,the serumlevel of NGF in COPD patients in acute exacerbation stage and stablestage were increased （30.3±11.7ng/L vs 7.29±3.4ng/L,17.2±8.5ng/L vs7.29±3.4ng/L,P＜0.01）, furthermore, the serum level of NGF in COPDpatients in acute exacerbation stage were elevated compared with instable stage（30.3±11.7ng/L vs 17.2±8.5ng/L,P＜0.05）. the significantnegative correlation was found between the NGF and the FEV₁%pre、FEV₁/FVC%, in acute exacerbation stage, rs=-0.73, rs=-0.72（P＜0.01）, while in stable stage, （rs=-0.65, rs=-0.68, P＜0.05）。 As a result, we can suggest that NGF play a critical role in theairflow limitation in COPD through the whole course of disease.2. COPD Model establishment.（1） 30 Wistar rats were divided into control and COPD grouprandomly, The COPD model was established by expose the rat to thecigarette and PPE intratracheal injection, lung function tests wasperformed by lung function instrument. （2） the lung tissues pathologicchanges were detected by HE staining. （3）The total cell count anddifferential cell count in the bronchoalveolar lavage fluid （BALF） wereoperated.Results showed: （1） in comparsion with the normal group, PEF（23.61±2.67 ml/s vs 30.96±2.88 ml/s）、FEV_0.3 （3.49±0.42 ml vs4.86±0.36 ml） and FEV_0.3/FVC [（62.25±4.84）% vs （86.68±6.12）%]decreased significantly （P＜0.01）; MLI （136.92±16.71μm vs 36.70±4.48μm） increased significantly, MAN （11.07±0.26/μm² vs 24.40±2.83/μm²） decreased significantly （P＜0.01）; The BALF total cell count（10⁴/ml） （23.93±3.52 VS 15.27±2.40） and the BALF neutrophilsincreased significantly （%） （7.56±1.47 VS 1.80±0.68）（P＜0.01）.In study content 2, presenting with airway inflammation andemphysema, obstructive ventilation functional impairment,we createdCOPD model successfully.3. nerver growth factor roles and its mechanism of regulatingairway neurogenic inflammation in COPD.To explore the roles and mechanisms of NGF in COPD and confirmour hypothesis,an anti-NGF group besides the COPD group and thenormal control group was established meanwhile. Lung function、HEstaining and the total cell count and differential cell count in the BALFwere also performed. NGF Quantitative analysis in the BALF weredetermined by ELISA.furthermore,a series of experimental methods were adopted.the localization and half quantitative analysis of NGF in thelungs and dorsal root ganglion（DRG） in the three groups were detectedrespectively by immunohistochemistry assay and Western-blot.NGFmRNA expression in lungs and DRG in the COPD and the control groupwere determined by reverse transcription-polymerase chain reaction（RT-PCR）,the changes of SP or NK-1R were investigated byimmunohistochemistry assay or in situ hybridization in the DRG or lungsin the three groups.Results showed: （1） compared COPD group with the normal group,the levels of NGF in the BALF were significantly enhanced （ng/L）（187.18±64.76 vs 75.13±20.44, P＜0.001）; the expression of NGF in thelungs and DRG increased significantly in the lungs and DRG inimmunohistochemistry average gray scale （104.23±7.74 vs153.50±8.02, 108.95±7.02 vs 171.03±8.14） （P＜0.01）; NGF increasedin Western blot assay（400.4±51.6ng/L vs 198.5±46.2ng/L,318.8±52.6ng/L vs 144.2±32.7ng/L）（P＜0.01）; NGF mRNA are enhancedin the lungs （82.07±7.89 vs 51.80±8.34） （P＜0.01）, on the contrary, nodifference were found in the DRG （56.67±6.28 vs 51.87±7.01） （P＞0.05）;NK-1R increased in the lungs in immunohistochemistry （average grayscale 101.33±6.31 vs 157.67±8.15） （P＜0.01）, while SP increased in theDRG in immunohistochemistry （average gray scale 103.40±7.08 vs167.06±7.83） （P＜0.01）, no SPmRNA change found in the lungs inimmunohistochemistry （165.53±7.30 vs 168.07±7.05） （P＞0.05）,whereas increased in the DRG （103.58±9.63 vs 173.37±12.83）（P＜0.01）. （2） compared the anti-NGF group with the COPD group:NGF wre decreased in the lungs and DRG both in immunohistochemistry（average gray scale 130.78±6.35 vs 104.23±7.74, 135.68±7.13 vs108.95±7.02） and Western blot （297.9±43.3ng/L vs 400.4±51.6ng/L,222.3±46.6ng/L vs 318.8±52.6ng/L） （P＜0.01）; meanwhile NK-1R decreased significantly in the lungs in immunohistochemistry （averagegray scale 138.20±7.99 vs 101.33±6.31） （P＜0.01）; also SP decreased inthe DRG in immunohistochemistry （average gray scale 138.00±9.32 vs103.40±7.08） （P＜0.01）; correspondingly SPmRNA decreased in theDRG in immunohistochemistry （average gray scale 143.21±11.30 vs103.58±9.63） （P＜0.01）; in BALF, the total cell count and the neutrophilratio decreased （17.13±3.94 vs 23.93±3.52,4.65±0.80 vs7.56±1.47）（P＜0.01）; in lung function,PEF, FEV0.3 and FEV0.3/FVC（27.88±2.98 vs 23.61±2.67, 3.93±0.25 vs 3.49±0.42,71.48±5.38 vs62.25±4.84）were elevated significantly （P＜0.05）, morphologically, MLIdecreased significantly （101.88±19.67 vs 136.92±16.71,P＜0.01）. （3）the significant correlation was found between the NGFmRNA in thelungs and the SPmRNA in the DRG inthe COPD group （rs=-0.81,P＜0.001）.we can conclude that NGF contribute to COPD developing.We canalso conclude that NGF may modulate the neurogenic inflammation inCOPD through down regulation the synthesis and secretion ofSP, anti-NGF can amend the pathological change and relieve the airflowlimitation.In conclusion, our current studies showed: NGF can regulateneurogenic inflammation through adjust the SP synthesis and release ofsensory neuron;NGF contribute to the airflow limitation in COPD;dealing with anti-NGF can bring SPmRNA and SP protein downregulation,palliate the airway inflammation in COPD. After the studycontents, we can clarify our hypothesis that NGF serving as an importantregulator in neurogenic inflammation in COPD.更多还原