【作者】 张程；

【导师】 陈平；

【作者基本信息】 中南大学 ， 呼吸内科， 2007， 博士

【摘要】 目的探讨肿瘤坏死因子-α（TNF-α）在大鼠慢性阻塞性肺疾病（COPD）发病中的作用及重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白（rhTNFR：FC）的干预影响。方法48只大鼠随机分为4组：正常对照组、COPD组、重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白干预组及假干预组。单纯吸烟法建立COPD模型，干预组皮下注射rhTNFR：Fc进行干预，假干预组皮下注射空白模拟制剂。各组随机抽取半数大鼠以小动物肺功能测定系统测定肺功能。酶联免疫吸附法（ELISA）测定各组血清和支气管肺泡灌洗液（BALF）中的TNF-α浓度。支气管肺泡灌洗计数BALF中白细胞总数、中性粒细胞和巨噬细胞计数及其百分比，肺组织切片行苏木素-伊红染色观察形态学改变，并定量测定肺平均内衬间隔（MLI）、平均肺泡数（MAN），利用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记（TUNEL）技术对肺泡隔凋亡细胞的阳性率进行检测，ELISA检测BALF中血管内皮细胞生长因子（VEGF）含量，Western blot检测血管内皮细胞生长因子受体2（VEGFR-2）蛋白、基质金属酶-9蛋白（MMP-9）的表达，使用自动生化分析仪测定各组大鼠血清白蛋白含量。结果（1）COPD组0.3秒用力呼气量占用力肺活量比值(FEV_0.3／FVC)和呼气峰流速（PEF）较正常对照组和rhTNFR：Fc干预组降低（P＜0.05），但和假干预组比较差异无统计学意义（p＞0.05）。假干预组FEV_0.3／FVC、PEF较rhTNFR：Fc干预组降低（p＜0.05）。（2）COPD组血清中TNF-α浓度高于正常组（P＜0.05）、rhTNFR：Fc干预组（P＜0.05），和假干预组比较差异无统计学意义（p＞0.05）。假干预组血清中TNF-α浓度高于rh／NFR：Fc干预组，差异有显著性（p＜0.05）。COPD组BALF中TNF-α浓度高于正常对照组（P＜0.05），但和rhTNFR：Fc干预组、假干预组比较差异均无统计学意义（p＞0.05）。（3）COPD组BALF中白细胞总数、中性粒细胞计数及百分比高于正常组（P＜0.01，P＜0.05，P＜0.05）、rhTNFR：Fc干预组（P＜0.01，P＜0.05，P＜0.05，），和假干预组比较差异无统计学意义（p＞0.05）。假干预组BALF中细胞总数、中性粒细胞计数及百分比高于rhTNFR：Fc干预组（P＜0.01，p＜0.05，P＜0.05）。（4）COPD组MLI比正常对照组增高（p＜0.05），其MAN比正常对照组、rhTNFR：Fc干预组降低（P＜0.05），但MLI、MAN和假干预组比较差异均无统计学意义（p＞0.05）。假干预组MAN低于rhTNFR：Fc干预组（p＜0.05）。（5）COPD组肺泡隔细胞凋亡指数（AI）高于正常对照组、rhTNFR：FC干预组（P＜0.05），和假干预组比较差异无统计学意义（p＞0.05）。假干预组肺泡隔细胞AI高于rhTNFR：FC干预组，差异有统计学意义（P＜0.05）。（6）COPD组BALF中VEGF含量较正常对照组降低（P＜0.05），肺组织中VEGFR-2蛋白表达较正常对照组降低（P＜0.05），和假干预组比较差异无统计学意义（p＞0.05）。COPD组肺组织中MMP-9表达较rhTNFR：Fc干预组升高（P＜0.05）。（7）COPD组体重较正常对照组、rhTNFR：Fc干预组体重降低，（P＜0.01），和假干预组比较差异无统计学意义（p＞0.05）。COPD组血清白蛋白较正常对照组降低（p＜0.05），但与rhTNFR：Fc干预组和假干预组比较差异无统计学意义（p＞0.05）。（8） （COPD组+假干预组）血清中TNF-α浓度与FEV_0.3／FVC、PEF呈负相关（r=-0.67，F=-0.77，p＜0.05）。（COPD组+假干预组）BALF中TNF-α浓度与FEV_0.3／FVC、PEF呈负相关（r=-0.83，r=-0.76，p＜0.05）。COPD组大鼠血清TNF-α浓度与体重呈负相关（r=-0.75，p＜0.05），与血清白蛋白浓度呈负相关（r=-0.73，p＜0.05）。结论（1）TNF-α与吸烟大鼠COPD形成有关，并参与肺部病理变化、气道炎症形成、损害肺功能、导致营养不良，促进肺泡隔细胞凋亡。（2）VEGF含量降低和肺组织VEGFR-2表达下降可能和吸烟大鼠肺气肿形成有关。（3）rhTNFR：Fc通过抑制TNF-α对改善吸烟大鼠肺部病理、减轻气道炎症、延缓肺功能损害、降低肺泡隔细胞凋亡凋亡、改善营养状态具有一定作用。更多还原

【Abstract】 ObjectiveTo investigate the role of TNF-αand the influence of recombinant human tumor necrosis factor-Fc on pathogenesis of chronic obstructive pulmonary disease of rats.MethodsForty eight rats were randomly divided into a normal control group, a COPD group, a rhTNFR: Fc-interfered group and a sham interfered group. Rat models of COPD were established by exposure to cigarette smoking daily for 80 days. rhTNFR: Fc-interfered group was interfered with rhTNFR: Fc, the sham interfered group injected with certain praeparatum as control for rhTNFR: Fc-interfered group. Lung function was detected in half rats of every group, the levels of Tumor necrosis factor alpha（TNF-α）in serum and BALF measured with ELISA. Total cell counts, neutrophil counts and neutrophil proportion in BALF were examined. Lung tissure section stained by hematoxylin and eosin （HE） was observed to study the morphological alternations. Mean linear intercept （MLI） and mean alveolar numbers （MAN） were measured. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） methods were carried out to examined the percentage of positive cells and distribution of apoptotic cells. The concentration of VEGF in BALF was examined with ELISA, the expression of VEGFR-2 and MMP-9 measured by Western blot and levels of albulmin in serum measured by automatic biochemistry analyzer.ResultsCompared with the normal control and rhTNFR: Fc-interfered group, FEV_0.3/FVC and PEF were lower in COPD rats （P＜0.05）; Compared with rhTNFR: Fc-interfered group, FEV_0.3/FVC and PEF were lower in sham interfered group（P＜0.05）. The levels of TNF-αin serum were higher in COPD group than in the normal control （P＜0.05） and rhTNFR: Fc-interfered group （P＜0.05）. Significanct differences were found between the levels of TNF-αin serum of rhTNFR: Fc-interfered group and sham interfered group（P＜0.05）. The levels of TNF-αin BALF were higher in COPD group than in the normal control（P＜0.05）, but no significanct differences were found between the levels in COPD group and rhTNFR: Fc-interfered group. Total cell counts and neutrophil counts and neutrophil proportion in BALF were higher in COPD group than in the normal control and rhTNFR: Fc-interfered group （P＜0.01, p＜0.05）, and they were also higher in rhTNFR: Fc-interfered group than in sham interfered group （P＜0.01, p＜0.05）. MLI in COPD rats was higher than that in the normal control（p＜0.05） while MAN was decreased on the contrary（p＜0.05）, compared with normal control and rhTNFR: Fc-interfered group. Significanct differences were found between MAN in rhTNFR: Fc-interfered group and sham interfered group. The number of TUNEL＋cells in alveolar septa was significantly increased in COPD group as compared with normal control and rhTNFR: Fc-interfered group（P＜0.05）, so was it in the latter compared with sham interfered group.The concentration of VEGF in BALF of COPD group was significantly decreased as compared with normal control group, but no significanct differences were found between the concentration of COPD group and rhTNFR: Fc-interfered group （p＞0.05）. The expression of VEGFR-2 protein in COPD group was significantly decreased compared with normal control group while no differences existed between that in COPD group and rhTNFR: Fc-interfered group （p＞0.05）. The expression of MMP-9 was decreased in rhTNFR: Fc-interfered group compared with COPD group（P＜0.05）.The weight of COPD group was significantly decreased compared with normal control group and rhTNFR: Fc-interfered group （p＜0.01）. No differences were found between the weight of COPD group and the control group for interfered one （p＞0.05）, significant differences found between the weight of rhTNFR: Fc-interfered group and control group for interfered one （p＜0.01 ）. The levels of albulmin in serum of COPD group were significantly decreased compared with normal control group （p＜0.05）, but no differences were found among the levels of COPD group and rhTNFR: Fc-interfered group and control group for interfered one （p＞0.05）. Negative correlations were demonstrated between the levels of TNF-αin serum and BALF of （COPD group＋sham interfered group） and PEF and FEV_0.3/FVC（p＜0.05）. The levels of TNF-αin serum of COPD group revealed negative correlations to the weight （r=-0.75, p＜0.05） and the levels of serum albumin （r=-0.73, p＜0.05）.ConclusionsTNF-αrelates to the pathogenesis of COPD of smoking rats and causes pulmonary pathological changes, airway inflammation, decline of pulmonary function, apoptosis of alveolar septal cells and malnutrition in smoking rats. Decrease of VEGF and VEGFR may contribute to the pathogenesis of emphysema of smoking rats. rhTNFR: Fc may play certain role in the improvement of pulmonary pathological changes, reduction of airway inflammation, decrease of alveolar septal apoptosis, slowdown of pulmonary function impairment of COPD and improvement of nutritional status.更多还原