【作者】 顾宇彤；

【导师】 蔡映云； 瞿涤； 何礼贤； 朱蕾；

【作者基本信息】 复旦大学 ， 内科学， 2005， 博士

【摘要】 氧化应激增强、氧化抗氧化失衡在慢性阻塞性肺病(COPD)发病机制中的作用正受到越来越多的关注。但对急性发作期氧化应激与炎症反应之间的关系以及抗氧化剂对氧化抗氧化失衡和炎症反应的作用尚无一致的结论。本研究观察单个核细胞炎性细胞因子TNF-α和IL-8 mRNA表达与反应性氧核素(ROS)产生的关系以及抗氧化剂N—乙酰半胱氨酸(NAC)能否减少ROS产生从而抑制炎症反应。探讨COPD急性发作期氧化抗氧化变化以及与炎症反应的关系。研究分四部分:1.LPS刺激THP-1细胞后TNF-α和IL-8 mRNA表达和反应性氧核素产生以及N—乙酰半胱氨酸对其影响的研究。2.COPD急性发作期患者氧化抗氧化失衡与炎症反应关系的研究。3.LPS刺激COPD缓解期患者血单个核细胞后炎性细胞因子表达和反应性氧核素产生以及N—乙酰半胱氨酸对其影响的研究。4.慢性阻塞性肺病急性发作期患者呼出气冷凝水8—异前列腺素测定及其意义。 第一部分:LPS刺激THP-1细胞后TNF-α和IL-8 mRNA表达和反应性氧核素产生以及N-乙酰半胱氨酸对其影响的研究 目的:观测LPS刺激THP-1细胞后TNF-α和IL8 mRNA表达、反应性氧核素(ROS)产生以及抗氧化剂N-乙酰半胱氨酸(NAC)能否减少ROS产生从而抑制炎症反应。方法:体外培养THP-1细胞株,荧光微量平板法测定THP-1细胞不同时间(0、0.5、1、1.5、3、24和48小时)ROS产生,荧光微量平板法和流式细胞仪测定不同浓度LPS(20、40、60、80、100和120ng/ml)刺激THP-1细胞24小时后ROS产生以及不同浓度NAC(0.1、1、10和20mM)对其的影响。RT-荧光实时定量PCR测定不同浓度LPS刺激THP-1细胞后TNF-α和IL-8mRNA表达以及更多还原

【Abstract】 Part ⅠOxidative Burst Response and Expression of TNF-α和 IL-8 mRNA in THP-1 Stimulated by LPS and Effects of N-acetylcysteine on itObjective To observe Oxidative Burst Response and Expression of TNF-and IL-8 mRNA in THP-1 cell line stimulated by LipoPoly Saccharide (LPS) and the effect of N-Acetylcy steins(NAC) on it. Methods THP-1 cells were cultured in vitro oxidative burst response i.THP-1 measured as the production of Reactive Oxygen Species(ROS) was performed by a fluorescence microplate assay using dichloridehydrofluorescein diacetate(H2DCFDA) at 0,0.5,1,1.5,3,24 and 48 hour. 24 hours after incubated with different concentration of LPS (20,40,60,80,100 and 120ng/ml), with or without different concentration of NAC (0. 1, 1, 10 and 20mM),ROS was measured by fluorescence microplate assay and flow cytometry using H2DCFDA, and the expression of TNF- and IL-8 mRNA in THP-1 cell was performed by reverse transcription-fluorescence real time polymerase chain. (RT-flourescence realtime PCR). Results 1. THP-1 Cell Oxidative Burst Response increased linearly over the course of incubation up to 24 hour, then downtrend was observed up to 48 hour. 2. With augment of concentration of LPS, ROS and expression of TNF- and IL-8 mRNA in THP-1 cell increased, peaking at 80ng/ml, then descended. 3.Incubated with 80ng/ml LPS and different concentration of NAC for 24 hour, Oxidative Burst Response and Expression of TNF- and IL-8 mRNA in THP-1 cell wasinhibited. Conclusion Stimulated by LPS,oxidative burst response and expression of TNF- and IL-8 mRNA in THP-1 cell increased, and inhibited by NAC(>0.1mM).Part Ⅱ Oxidant/Antioxidant Imbalance and Inflammation in COPDObjective In order to study the relationship between Oxidant/Antioxidant Imbalance and Inflammation in chronic obstructive pulmonary disease (COPD), the expression of TNF- α and IL-8 mRNA and ROS in peripheral blood mononudear cell(PBMC) from aggravated patients with COPD, malondialdehyde (MDA) and total antioxidant capacity (TAC) in blood plasma, white blood cell(WBC) and neutrophil were investigated. Methods Venous blood sample were obtained in heparin anticoagulation tests from 15 aggravated COPD patients in hospital. PBMC were isolated from blood samples by differential centrifugation over Ficoll-Paque. PBMC were suspended in medium RPIM 1640,and incubated with LPS 80ng/ml for 24 hours, the expression of TNF- α and IL-8 mRNA were analyzed by RT-flourescence realtime PCR. Meanwhile ROS was performed by flow cytometry using H2DCFDA. White blood cell(WBC) and neutrophil were counted, MDA and TAC in blood plasma were measured. These indexes were repeated again in catabasis. 115 stable COPD patients and 15 no smoking donors were selected as control. Results Average and total ROS expressed as relative fluorescence unit(RFU) (718.5±353.7 and 66260.7 ±34181.1 RFU),the expression of TNF-α and IL-8 mRNA expressed as ratio of GAPDH in PBMC(0.785 ±0.383 and 0.188 ± 0.16/GAPDH), MDA(6.07 ±0.85nmol/ml) in blood plasma were higher during exacerbation stage than convalescence (218.7±102 and 21144.5±9485.4 RFU,0.056±0.050 and 0.027 ±0.016/GAPDH, 5.1±0.84 nmol/ml respectively, P<0.05) . there was no significant difference of ROS positive gate in PBMC and TAC in blood plasma between in exacerbation stage(91.3 ± 8.3% and 13.28 ± 3.88 u/ml) and convalescence stage(97.6 ±2.2 % and 13.83 ± 3.76 u/ml, p>0.05). 2.In the exacerbated patients with COPD,A significant and positive correlations was present between the expression of TNF- a mRNA and IL-8 mRNA , MDA in blood plasma, WBC and neutriphil counts and total ROS(r=0.78、 0.62、 0.72、 0.70 and 0.71 respectively, p<0.05).There is no significant correlations between the total ROS and TAC in blood plasma。 3. ROS positive gate and total RFU in PBMC of the COPD patients in convalescence stage (97.6 ±2.2% and 21144.5 ± 9485.4 RFU) were higher than stable (90.3 ± 9.1 % and 15083.4 ± 3957.3 RFU,p<0.05) . There is no significant difference of mean ROS ,expression of the TNF- α mRNA and IL-8 mRNA, MDA and TAC in blood plasma between the COPD patients in convalescence stage and stable stage (P>0.05 ) .4. Mean and total ROS, MDA in blood plasma in stable COPD patients (172.05 ± 58.2 RFU, 21144.5 ± 9485.4 RFU and 4.79 ± 0.13 nmol/ml respectively ) were significantly higher than no smoking healthy people (133.4 ± 34.4 RFU, 11151.9 ± 2867.5 RFU and 2.6 ± 0.52 nmol/ml respectively, P<0.05) . There was no significant difference of ROS positive gate, expression of TNF- mRNA and IL-8 mRNA in PBMC and TAC in blood plasma between them(P>0.05). 5.TAC in the exacerbated COPD patients blood plasma(13.28 ± 3.88 u/ml) was significantly lower than no smoking healthy people(18.31±4.84 u/ml, p<0.05) . Conclusion In the exacerbated patients with COPD, Expression of genes for inflammatory cytokines and ROS in PBMC augment. The systemic Oxidative stress increases In clinical convalescence and stable COPD patients, the expression of genes for inflammatory cytokines in PBMC restores, the systemic Oxidative stress decreases , but higher than no smoking healthy people. The total antioxidant capacity in the exacerbated COPD patients was lower than no smoking healthy people. The systemic .Oxidative stress in the exacerbated COPD patients may be related with inflammatory reaction.Part ⅢOxidative Burst Response and Expression of genes forinflammatory cytokines in PBMC of stable COPD patientsstimulated by LPS and the effects of N-Acetylcysteine on itObjective To observe Oxidative Burst Response and Expression of TNF-a mRNA and IL-8 mRNA in PBMC of stable COPD patients stimulated by LPS and the effects of NAC on it. Further to study the relationship between oxidative stress and inflammation, and whether or not is antioxidant able to decrease oxidative stress, than relieve inflammatory reaction. Methods Venous blood samples were obtained in heparin anticoagulation tests from 10 stable COPD patients. PBMC were isolates from blood sample by differential centrifugation over Ficoll-Paque. PBMC were suspended in Medium(RPMI1640). Subjects were divided into 3 groups: no LPS group , LPS 80ng/ml group, and NAC:LPS 80ng/ml+NAC 1. OmM group. PBMC were harvested after a 24h incubation at 37℃ in 5% CO2. ROS was performed by flow cytoetry using H2DCFDA. The expression of TNF-α mRNA and IL-8 mRNA wasanalyzed by RT-flourescence realtime PCR. Results 1. There is no correlation between FEV1, FEV1/pred, FVC and ROS (p>0.05). 2. The expression of TNF- α mRNA and IL-8 mRNA, mean and total ROS were higher in PBMC of LPS group(0. 664 ± 0. 473, 0. 794 ± 0. 585, 536. 2 ± 247. 1 RFU and 44130. 1 d=16008. 3 RFU respectively) than control (0.240 ± 0.238,0.155 ± 0.139,340.6 ± 110.3 RFU and 31700.2 ± 10103.0 RFU respectively, p<0. 05) . There was no significant difference of ROS positive gate between group LPS and control (P>0. 05) . 3. In LPS group , A significant and positive correlation was present between the expression of TNF- a a mRNA or IL-8 mRNA and mean or total ROS respectively. There was no significant difference between ROS positive gate and the expression of TNF- a mRNA and IL-8 mRNA. 4. The expression of TNF-α mRNA and IL-8 mRNA , mean and total ROS in group NAC(0. 0119 ± 0. 0078,0. 0041 ± 0. 0020,99. 2 ± 62. 9 RFU and 9309. 5 ± 6349.7 RFU respectively, P<0.001) were obviously lower than LPS group (P<0. 05) . Conclusion LPS as an inflammatory stimulation induces the stable COPD patients PBMC expression of proinflammatory cytokines and oxidative Burst Response increasing , that were obviously inhibited by NAC.Part ⅣExhaled 8-Isoprostane as an In vivo Biomarker of Lung Oxidative Stress in Patients with exacerbated COPDObjective To quantity oxidative stress in the lungs in patients with exacerbated COPD, as reflected by 8-isoprostance concentration in exhaled breath condensate (EBC). Methods Breath condensate samples were collected in 15 patients with exacerbated COPD. Exhaled 8-isoprostance was measured by a specific enzyme immunoassay. Venous blood sample were obtained in heparin anticoagulation tests, from these patients MDA and TAC in blood plasma were measured, white blood cell and neutrophil were counted. These indexes were repeated again in catabasis.15 smoking and 15 no smoking healthy donors were selected as control. Results 1. 8-isoprostance concentration in EBC, blood plasma MDA, WBC and neutrophil更多还原