【作者】 张建全；

【导师】 钟小宁；

【作者基本信息】 广西医科大学 ， 呼吸内科， 2010， 博士

【摘要】 第一部分肺功能正常吸烟者和慢性阻塞性肺疾病患者肺组织中白细胞介素17的表达及意义目的探讨白细胞介素17(IL-17)在肺功能正常吸烟者和慢性阻塞性肺疾病(COPD)患者肺部炎症反应发生机制中的作用。方法将需手术治疗的周围型肺癌患者分为肺功能正常非吸烟组(NS组,10例)、肺功能正常吸烟组(S组,13例)、吸烟COPD稳定期组(COPD组,10例)。于手术切除标本选取距肺癌病灶5～8cm处的新鲜正常肺组织,酶联免疫吸附试验(ELASA)检测各组肺组织匀浆IL-17含量,HE和维多利亚蓝+范吉逊染色检测各组平均肺泡面积、小气道总病理积分和肺腺泡肌型动脉(MA)管壁厚度,免疫组织化学方法检测IL-17+及CD4+、CD8+T细胞在肺泡壁、小气道壁及MA管壁的表达,分析IL-17表达与CD4+、CD8+T细胞以及与肺实质病理改变和肺功能指标的相关关系。结果NS组、S组和COPD组肺组织匀浆IL-17含量分别为6.1(3.7～12.4)、9.7(3.5～69.7)和22.7 (7.0～114.4) pg/mg, COPD组明显高于明显NS组(P=0.000),COPD组明显高于S组(P=0.036),S组明显高于NS组(P=0.042)；平均肺泡面积分别为(50708±14125)、(106517±13851)和(152344±43783)μm2,小气道总病理积分分别为(49±10)、(101±34)和(163±36)分,MA管壁厚度分别为(119±11)、(139±25)和(172±28)μm,S组和COPD组均明显高于NS组(P<0.05,P<0.01),COPD组明显高于S组(P<0.05, P<0.01)。IL-17主要表达于肺实质浸润的炎症细胞,S组和COPD组肺泡壁、小气道壁及MA管壁IL-17+T细胞均明显多于NS组(均尺0.01),COPD组明显多于S组(均P<0.05)。IL-17表达与平均肺泡面积(r=0.561)、MA管壁厚度(r=0.682)、小气道病理总积分(r=0.425)呈显著正相关(均P<0.05)。肺泡壁、小气道壁和MA管壁中IL-17表达与CD4+、CD8+细胞呈显著正相关(P<0.05,P<0.01)。肺组织匀浆IL-17含量与第1秒用力呼气容积占预计值百分比呈显著负相关(r=-0.471,P=-0.006)。结论IL-17在肺功能正常吸烟者和COPD患者肺组织中表达增高,与肺CD4+、CD8+T细胞及肺组织破坏、气道炎症、肺动脉重构和气流受限密切相关,提示IL-17在吸烟者和COPD患者肺部炎症反应发生机制中有促炎作用。第二部分辅助T细胞17在肺功能正常吸烟者及COPD患者肺部组织中的活化、分布及作用目的观察肺功能正常吸烟者和COPD患者肺组织是否存在活化的辅助T细胞(Th)17即CD4+IL-17+细胞,其活化程度以及与IL-17之间的相关关系,探讨肺实质中IL-17是否来源于Th17细胞。分析CD4+IL-17+细胞在肺部的表达及分布并与肺部病理、肺功能指标改变之间做相关分析关系,探讨Th17细胞在COPD炎症反应中的作用。方法实验病例入选及分组同第一章。荧光定量聚合酶链式反应(PCR)方法检测肺组织匀浆Th17细胞特异性转录因子ROR gamma t(维甲酸相关孤独受体γt)的RNA表达水平,蛋白质印迹(Westerm blot)方法检测肺组织匀浆ROR gamma t蛋白表达水平,免疫荧光双标方法检测Th17细胞在肺泡壁、小气道壁、肺小动脉壁的表达。分析肺组织匀浆中ROR gammat RNA表达水平与IL-17蛋白水平及FEV1占预计值百分比相关性；分析肺组织中Th17细胞与肺气肿、小气道病理总积分、肺小动脉管壁厚度,及与IL-17+、CD4+T淋巴细胞之间相关性。结果(1)荧光定量PCR结果显示,肺组织匀浆ROR gammat的mRNA表达水平,在NS组为(2.685±0.886),S组(8.564±1.419),COPD组(10.158±1.574),三组间两两比较有显著性差异(P<0.01或P<0.05)以COPD最为显著。(2) Western blot结果显示肺组织匀浆ROR gamma t蛋白表达水平在NS组(0.369±0.094),S组(0.614±0.241),COPD组90.886±0.184),三组间两两比较有显著性差异(P<0.01或P<0.05),以COPD组最为显著。(3)免疫荧光双标结果显示,CD4+IL-17+细胞在肺泡壁的表达,S组为(25±7)个／cm,COPD组为(27±4)个／cm较NS组(14±5)个／cm显著性增强(均P<0.01),但COPD组与S组比较无显著性差异(P>0.05)。在小气道壁的表达,COPD组为(220[81,422])个／mm2较S组(121[22,170])个／mm2及NS组(54[16,258])个／mm2表达增强(P<0.05),S组较NS组表达有增强的趋势,但无显著性差异(P>0.05)。NS组、S组、COPD组三组在血管壁的表达有增强的趋势但无显著性差异(均P>0.05)。(4)肺组织匀浆中ROR gammat RNA与FEV1占预计值百分比呈显著负相关(r=-0.643, p= 0.000), ROR gammat蛋白含量与FEV1占预计值百分比呈显著负相关(r=-0.539,p=0.002),肺组织IL-17蛋白含量与ROR gammat RNA含量呈显著正相关(r=0.678,p=0.000)。(5)肺泡壁中CD4+IL-17+细胞数与平均肺泡面积呈显著正相关(r=0.738,p=0.000),在气道壁中Th17细胞数与气道总病理评分呈显著正相关(r=0.476,p=0.034),在MA管壁中的Th17细胞数与血管壁厚度无显著性相关(r=0.355,p=0.125)。(6)肺泡壁中CD4+IL-17+细胞数与IL-17+、CD4+T淋巴细胞之间相关系数(分别r=0.584,0.646,均P<0.01),气管壁中CD4+IL-17+细胞数与IL-17+、CD4+T淋巴细胞之间相关系数(分别r=0.510,0.518,均P<0.05),肺小动脉壁CD4+IL-17+细胞数与IL-17+淋巴细胞之间相关系数(r=0.517,P<0.05),与CD4+T淋巴细胞无显著性相关(r=0.200,p=0.399)。结论Th17细胞特异性转录因子ROR gammat mRNA及蛋白水平在正常吸烟者的肺组织已经开始增高,COPD患者更为显著,并与IL-17蛋白含量密切相关；Th17细胞表达增强并与IL-17+细胞、CD4+T淋巴细胞及肺组织破坏、气道和肺动脉炎症与重构、气流受限密切相关。结果提示CD4+T淋巴细胞参与吸烟者和COPD患者肺部炎症免疫反应,肺组织中Th17处于增殖活化状态,并分泌IL-17参与并促进肺部炎症反应。第三部分Th17细胞及相关因子参与肺功能正常吸烟者和COPD患者肺部炎症的相关机制目的观察Th17细胞活化的上游因子IL-23受体(IL-23R), CC趋化因子受体6(CCR6)及其分泌的相关因子IL-17、IL-21在肺组织的表达及意义,探讨Th17细胞在COPD肺部炎症中活化及放大炎症反应的可能机制。方法实验病例入选及分组同第一章。肺组织蜡块病理切片免疫组化技术检测肺泡壁1L-17, IL-23R, CCR6的表达,并分析三者之间及与Th17细胞之间的相关性。荧光定量PCR检测肺组织匀浆IL-21mRNA、1L-17mRNA含量,并分析二者之间,以及与RORγtmRNA和FEV1的相关关系。分析肺泡壁、气道壁、肺动脉壁Th17细胞与CD8+T细胞的相关性。结果(1)在肺泡壁中,NS组、S组、COPD组IL-23R+细胞数分别为(46±7)个／cm, (60±15)个／cm, (65±12)个／cm,S组和COPD组与NS组比较,有显著性差异(P<0.05或P<0.01),COPD组较S组有增高趋势,但无显著性差异(P>0.05)。在肺泡壁中,NS组、S组、COPD组CCR6+细胞数分别为(42±6)个／cm, (56±8)个／cm, (62±8)个／cm,S组和COPD组与NS组比较,有显著性差异(P<0.05或P<0.01),COPD组较S组有增高趋势,但无显著性差异(P>0.05)。(2)荧光定量PCR检测结果显示,肺组织匀浆中,NS组、S组、COPD组三组的IL-21mRNA分别为(4.784±2.282),(46.312±15.463),(84.804±21.884),三组间两两比较有显著性差异(均P<0.01),COPD组表达增高最明显。NS组、S组、COPD组三组的IL-17mRNA分别为(3.121±1.262),(26.434±7.529),(44.929±15.303),三组间两两比较有显著性差异(P<0.01),COPD组表达增高最明显。(3)肺泡壁中,CD4+IL-17+细胞数与IL-17+细胞数、IL-23R+细胞数、CCR6+细胞数有显著正相关(r分别为0.584,0.422,0.670,P<0.01或P<0.05)。IL-17+细胞数与IL-23R+细胞数无显著性相关(r=0.302,p=0.105)。IL-23R+细胞数与CCR6+细胞数有显著正相关(r=0.572,p=0.001),IL-17+细胞数与CCR6+细胞数有显著正相关(r=0.508,p=0.004)。(4)肺泡壁中CD4+IL-17+细胞数与CD8+T淋巴细胞之间相关系数为(r=0.664,P<0.01),气管壁中CD4+IL-17+细胞数与CD8+T淋巴细胞之间相关系数为(r=0.491, P<0.05),肺小动脉壁CD4+IL-17+细胞数与CD8+T淋巴细胞之间相关系数为(r=0.518,P<0.05)。(5)肺组织匀浆中IL-21mRNA与RORγtmRNA含量呈显著正相关(r=0.757,p=0.000),R0RγtmRNA与IL-17mRNA含量呈显著正相关(r=0.802,p=0.000),IL-21mRNA与IL-17mRNA含量呈显著正相关(r=0.746,p=0.000)。(6)肺组织匀浆中IL-21mRNA、1L-17mRNA与FEV1均呈显著负相关(r分别为-0.694,-0.725,均p=0.000)。结论肺功能正常吸烟者和COPD患者肺泡壁中IL-17、IL-23R、CCR6表达显著增高并与Th17细胞数呈显著正相关,在肺泡壁、气道、肺动脉中Th17细胞数与CD8+淋巴细胞均有显著正相关,Th17细胞相关因子IL-21、1L-17在肺组织匀浆中RNA转录水平与RORγtmRNA之间均呈显著正相关,与气流受限密切相关。结果提示肺功能正常吸烟者和COPD患者中Th17通过CCR6及IL-23途径趋化浸润到肺实质并增殖活化,分泌工L-17、IL-21等细胞因子,调节中性粒细胞及CD8+T淋巴细胞共同发挥致炎及放大炎症的作用。更多还原

【Abstract】 PARTⅠInterleukin-17 Expression and Significance in Normal Lung Function Smokers and Chronic Obstructive Pulmonary Disease PatientsObjective To study the pulmonary inflammatory mechanism of interleukin 17 (IL-17) in the normal lung function smokers and chronic obstructive pulmonary disease (COPD) patients.Methods The peripheral lung cancer patients which need surgical treatment were divided into normal lung function non-somking group (NS group, 10 cases), normal lung function and smoking group (S group,13 cases), smoking with stable COPD group (COPD group,10 cases). Select the fresh normal lung tissue from the surgical specimens whice is 5cm over from the lung cancer resection, then detected the lung tissue IL-17 levels with Enzyme-linked immunosorbent assay (ELISA), the average alveolar area, the total integration of small airway pathologyacinar, and the pulmonary muscular artery (MA) wall thickness by HE and Victoria blue-van Gieson’s stain, the IL-17+cells and CD4+, CD8+lymphocytes in the alveolar walls, small airways and lung muscular artery (MA) by immunohistochemistry. Analyze the relationships between IL-17 level, pathological morphology of lung parenchyma, small airway, pulmonary artery reconstruction and pulmonary function.Results The IL-17 levels in lung tissue of NS group, S group and COPD group were 6.1 (3.7～12.4),9.7 (3.5～69.7) and 22.7 (7.0～114.4) pg/mg respectively. The COPD group was significantly higher than the NS group (P =0.000) and the S group (P=0.036), the S group was significantly higher than the NS group (P=0.042). The average alveolar area were (50708±14125), (106517±13851) and (152344±43783)μm2,the total integration of small airway pathology were(49±10), (101±34) and (163±36), the MA wall thickness were (119±11),(139±25) and (172±28)μm. The S group and the COPD group were significantly higher than the NS group (P<0.05, P<0.01), and the COPD group was significantly higher than the S group (P<0.05, P<0.01).IL-17 was mainly expressed in the lung infiltration of inflammatory cells, IL-17 of the S group and COPD group in the alveolar walls, small airway wall and the MA wall were significantly higher than the NS group, and the COPD group was significantly higher than NS group (P<0.05). IL-17+cells were positively correlated with the average alveolar area in the lung parenchyma (r=0.561, P<0.01), the pulmonary artery wall thickness in the MA(r=0.682, P<0.01), and the pathological score in the small airways (r=0.425, P<0.05). IL-17+cells of the lung parenchyma, small airways and the MA, were positively correlated with CD4+and CD8+ lymphocytes in the lung(P<0.05, P<0.01). The levels of IL-17 in lung homogenate tissue showed a negative correlation with the FEV1 percentage of predicted value (r=-0.471, P=0.006).Conclusions The IL-17 was increased in the lung tissues of normal lung function smokers and COPD patients, and was closely related to CD4+and CD8+lymphocytes in the lung, lung parenchyma destruction, pulmonary inflammation, pulmonary artery reconstruction and airflow limitation. All of these suggested that IL-17 playing an important pro-inflammatory role in COPD.PARTⅡThe Activation, Distribution and Contribution of Th17 cell in Normal Lung Function Smokers and Chronic Obstructive Pulmonary Disease PatientsObjective To observe the normal lung function smokers and COPD patients lung tissue whether they are supporting the existence of activated Th 17 cells, and the activation relationship with IL-17,then explore the lung parenchyma IL-17 whether it derived from Thl7 cells, and analyzed CD4+IL-17+cells expression and distribution in the lung, the correlation with the pulmonary pathology and lung function changes, explored the role that Th17 cell play on the COPD Pulmonary inflammation.Methods Test cases selected and grouped as the first chapter. Measured lung homogenates RORγt (retinoic acid receptor-related orphanγt) mRNA expression levels by Quantitative PCR, detected lung tissue ROR gamma t protein expression levels by Western blot, detected Th17 cells expression in the alveolar walls, small airway wall, pulmonary arterioles by double immunofluorescence. The relevance of ROR gammat RNA expression in the lung tissues was analyzed between IL-17 protein levels and FEV1 percentage of predicted value. The correlationship of Th17 cells in lung tissue was Analyzed with emphysema, small airway pathology total score, pulmonary artery wall thickness, IL-17+cells and CD4+T lymphocytes.Results (1)The results of Quantitative PCR showed that, the mRNA expression level of lung tissue ROR gammat in the NS group was (2.685±0.886), the S group was (8.564±1.419), the COPD group was (10.158±1.574), compared with each group, the three groups had significant difference (P<0.01 or P<0.05), and the COPD was the most significant. (2) The result of Western blot showed that the ROR gammat protein expression levels in the lung tissue of the NS group was (0.369±0.094), the S group was (0.614±0.241), the COPD group was (90.886±0.184), compared with each other the three groups had significant differences (P<0.01 or P<0.05), and the COPD group was the most significant. (3)The result of Immunofluorescence double labeling showed that the expression of CD4+IL-17+cells in the alveolar wall, the S group was (25±7) cells/cm,the COPD group was (27±4) cells/cm compared with NS group (14±5) cells/cm, the COPD group had a significantly increased (all P<0.01), but the COPD group and the S group showed no significant difference (P> 0.05).In small airway wall the expression of COPD group (220 [81,422])cells/ mm2 was stronger than the expression of S group (121 [22,170]) cells/mm2 and NS group (54 [16,258]) cells/mm2 (P<0.05), the tendency which the expression of CD4+IL-17+cells was more in S group than that in NS group exist but without significant difference (P>0.05). (4) The ROR gammat RNA in the Lung tissues had a significant negative correlation with FEV1 percentage of the predicted value (r=-0.643, p= 0.000), the ROR gammat protein content and the FEV1 percentage of the predicted value was significant negatively correlated (r =-0.539, p= 0.002),but the lung tissue IL-17 protein and the ROR gammat RNA content was significant positively correlated (r=0.678,p=0.000). (5) The CD4+IL-17+cells in the alveolar wall was significant positively correlated with the mean alveolar area (r= 0.738, p= 0.000), in the airway wall the Th17 cells and airway total pathology score was significantly correlated (r= 0.476, p= 0.034), but the Th17 cells of MA wall showed no significant correlation with vascular wall thickness (r=0.355, p=0.125).(6) In alveolar wall, the CD4+IL-17+cells and the IL-17+lymphocyte cells show positive correlation (r= 0.584, P<0.01), and the CD4+lymphocyte cells (r= 0.646, P<0.01) In small airway wall, the CD4+IL-17+cells and the IL-17+lymphocyte cells show positive correlation (r= 0.510, P<0.05), and the CD4+lymphocyte cells (r= 0.518, P<0.05). In the pulmonary artery wall, the CD4+IL-17+cells and the IL-17+lymphocyte cells show positive correlation (r= 0.517,P<0.05).but no significant correlation with the CD4+lymphocyte cells (r= 0.200, p= 0.399)Conclusions The Th17 cell-specific transcription factor ROR gammat mRNA and protein levels in lung tissue of normal smokers has begun to increase,and the COPD patients was more pronounced and closely related with IL-17 protein.Th17 cells and IL-17+cells, CD4+T lymphocytes were closely related to lung tissue destruction, airway and pulmonary inflammation and remodeling, airflow limitation. The results suggest that Th17 in the lung tissue of smokers and COPD patients was in the proliferation and activation state, which secreted the IL-17 participate and promote the lung inflammation. PART IIIThe Pulmonary Inflammation Mechanism of Th17 Cells and Related Factors Involved in Normal Lung Function Smokers and COPD PatientsObjective To investigate the expression and significance of upstream factor IL-23R and CCR6 which was activated by the Thl7 cells, and related factors IL-21 and IL-17 in lung tissue, and explored Thl7 activation and the possible mechanism of inflammatory amplification.Methods Test cases selected and grouped as the first chapter. Detected the expression of alveolar wall IL-17, IL-23R and CCR6 by immunohistochemical, analyzed each other relationship among IL-17, IL-23R and CCR6, and their correlation between Th17 cells. Detected the IL-21 mRNA, 1L-17mRNA content in the lung tissue by quantitative PCR, and analyzed the relationship between the two, as well as the correlation between RORγtmRNA and FEV1. Analyzed the correlation between Thl7 cells and CD8+T cells in alveolar wall, small airway wall and pneumono-arteriola wall.Results (1) IL-23R+cells in alveolar wall of the NS group, S group and COPD groups were (46±7), (60±15) and (65±12) cells/cm respectively, the S group and the COPD group compared with the NS group, there were significant differences (P<0.05 or P<0.01), COPD group tended to increase when compared with S group, but had no significant difference (P> 0.05). In the alveolar wall, the CCR6+cells of the NS group, S group and COPD group were (42±6), (56±8) and (62±8) cells/cm respectively, and there were significant differences when the S group and the COPD group compared with the NS group (P<0.05 or P<0.01), the COPD group were in the trend of increasing than the S group, but had no significant difference (P>0.05). (2) Quantitative PCR results showed that, IL-21mRNA in NS group, S group, COPD group were respectively (4.784±2.282), (46.312±15.463) and (84.804±21.884) in lung homogenates, compared with each other in the three groups,there were significant difference (all P<0.01), and the expression of COPD group increased most obvious.The IL-17mRNA in NS Group, S Group and COPD group were respectively (3.121±1.262), (26.434±7.529), (44.929±15.303), compared with each among the three groups there were significantly different (all P<0.01), and the expression of COPD group increased most obvious. (3)In the alveolar wall, CD4+IL-17+cells had a significant positive correlation with IL-17+cells, IL-23R+cells and CCR6+cells (r=0.584,0.422, 0.670, P<0.01 or P<0.05). IL-17+cells had no correlation with IL-23R+cells (r =0.302, p=0.105). IL-23R+cells and CCR6+cells had a significant positive correlation (r= 0.572, p= 0.001),and IL-17+cells and CCR6+cells had a significant positive correlation too (r=0.508, p=0.004). (4) In the alveolar wall, the correlation coefficient between CD4+IL-17+cells and CD8+T lymphocytes was (r=0.664, P<0.01); in the airway wall, the correlation coefficient between CD4+IL-17+cells and CD8+T lymphocytes was (r= 0.491, P<0.05); in the pulmonary artery wall, the correlation coefficient between CD4+ IL-17+cells and CD8+T lymphocytes was (r= 0.518, P<0.05). (5)In the lung homogenate, IL-21mRNA and RORγt mRNA content had a significant positive correlation (r=0.757,p=0.000), RORγt mRNA and IL-17mRNA content had a significant positive correlation (r=0.802,p=0.000), IL-21mRNA and IL-17mRNA content had a significant positive correlation (r= 0.746, p= 0.000). (6) In the lung tissue, IL-21mRNA, IL-17mRNA and FEV1 showed a significant negative correlation (r=-0.694,-0.725, respectively, all p=0.000).Conclusions In the alveolar wall of normal lung function smokers and COPD patients, the expression of IL17, IL-23R and CCR6 were significantly increased, and showed a significant positively relationship with Th17 cells. In the alveolar wall, airway and pulmonary artery, there was alse a significant positive correlation between Th17 cells and CD8+lymphocytes.The RNA transcription of Th17 cell-related factor IL-21, IL-17 in lung homogenates showed a significant positive correlation between RORγtmRNA, and was closely related with airflow limitation. The results suggest that in the normal lung function smokers and COPD patients, Th17 chemotactically invaded the lung parenchyma to proliferated and activated through CCR6 and IL-23,secreted IL-17 and IL-21 cytokines, regulating neutrophils and CD8+T lymphocytes, cause and enlarge the inflammation.更多还原