莊淳宇Chen, Chia-Yang;Sung, Fung-Chang;Chuang, Chun-Yu臺灣大學:環境衛生研究所黃依齡Huang, Yi-LingYi-LingHuang2010-05-072018-06-302010-05-072018-06-302009U0001-2312200814310700http://ntur.lib.ntu.edu.tw//handle/246246/181426吸煙過程中產生之活性氧化物種(reactive oxidative species; ROS)會造成呼吸道上皮細胞氧化傷害，促使細胞修復。持續性的細胞修復會使呼吸道重塑(airway remodeling)和不可逆的氣道受限(airflow limitation)。硫醇氧化還原蛋白(thioredoxin; TRX)為氧化還原蛋白，具有清除ROS特性進而減少氧化壓力。本研究目的欲瞭解TRX如何調控香菸暴露所導致之氧化傷害及其對於呼吸道重塑因子(airway remodeling factor)和細胞傳遞訊息(signal transduction)之影響。研究分別以人類呼吸道細胞和具有大量表現TRX呼吸道細胞，暴露不同劑量和時間的香菸萃取物，進而探討TRX參與調控呼吸道重塑因子表現之情形。結果顯示當人類呼吸道上皮細胞受到高濃度香菸暴露時，會產生內生性ROS和使TRX基因表現下降而導致細胞凋亡(apoptosis)；呼吸道上皮細胞暴露低濃度香菸則增加TRX基因表現。此外，香菸暴露增加重塑因子基因如transforming growth factor (TGF)-β1、epidermal growth factor receptor (EGFR)和cyclin-dependent kinase inhibitor (p21)之表現量，但抑制matrix metalloproteinases (MMP)-9。量表現TRX之呼吸道上皮細胞暴露香菸時，能減少ROS產生，並透過apoptosis signal regulating kinase (ASK)-1去活化及抑制 p38和c-Jun N-terminal kinase (JNK)表現，使細胞免於走向凋亡。呼吸道上皮細胞以TGF-β1抗體中和(neutralization)後再暴露香菸，TRX表現量受到抑制，使得MMP-9、EGFR和p21基因表現下降。此結果顯示TRX受到TGF-β1調控，進而使MAPK及MMP-9蛋白去活化。本研究證實轉殖的TRX具有氧化還原能力，且當TRX大量表現時會減少香菸暴露所造成之氧化傷害。此研究結果得知TRX抵禦氧化傷害之基因調控機制，可提供後續肺部損害相關研究之參考。Cigarette smoke (CS) generates reactive oxygen species (ROS) that produces oxidative damage to bronchial epithelial cells. Prolonged repair responses lead to airway remodeling and irreversible airflow limitation. Cigarette smoke may cause bronchial epithelium damage and orchestrate airway remodeling resulting from activation of epithelial repair and mediator release. Thioredoxin (TRX) has anti-oxidation capability against oxidative stress, and involves in cell growth and differentiation. The aim of this study was to investigate how TRX mediates CS-induced oxidative damage and modulated any onset/progression of airway remodeling and its downstream mitogen-activated protein kinase (MAPK) in human airway epithelial cells.ormal human epithelial cells (BEAS-2B) and thioredoxin over-expressing cells were used for various experiments including different doses and periods of CS exposure. Results of this study showed that high-dose CS extract stimulated ROS generation and decreased TRX expression resulting in apoptosis of BEAS-2B cells. Otherwise, BEAS-2B cells exposure to low-dose CS increased TRX expression. Additionally, CS exposure interfered with gene expression of remodeling factors such as activation of the transforming growth factor (TGF)-β1, epidermal growth factor receptor (EGFR) and cyclin-dependent kinase inhibitor (p21), but attenuated of matrix metalloproteinases (MMP)-9. EAS-2B cells over-expressing TRX suppressed CS-induced apoptosis through reduction of ROS generation, inactivation of apoptosis signal regulating kinase (ASK)-1, and attenuation of p38 and c-Jun N-terminal kinase (JNK). After being neutralized with anti-TGF-β1 antibody, TRX over-expressing cells exposed CS decreased gene expression of MMP-9, EGFR and p21. It suggests that TGF-β1 regulated TRX expression may further prevent the underlying CS-induced airway remodeling through MAPK activation and MMP-9 augmentation. This study demonstrated that the transfected TRX gene had redox capacity to reduce CS-induced oxidative damage. The mediating mechanism of TRX against CS-induced damage can be applied in further preventive and therapeutic studies.目錄錄 I表目錄 III要 1bstract 3bbreviation 5hapter 1 Introduction 6.1 Cigarette smoke (CS) causes lung diseases 6.2 Oxidative stress 7.3 Reactive oxidative species (ROS) induces inflammation 8.4 Antioxidation systems 9.4.1 Thioredoxin (TRX) 9.4.2 TRX and human diseases 10.4.3 TRX and oxidative stress 11.4.4 TRX and transcription factors 12.5 Mitogen-activated protein kinases (MAPKs) 12.6 TRX and MAPKs 14.7 Airway remodeling factors- TGF-β1, MMP-9, p21, EGFR 15.8 Study purpose 17hapter 2 Material and Method 20.1 Cell preparation 20.2 Extraction and exposure of CS 20.3 MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay 23.4 Plasmid construction of TRX gene tagged flag 23.5 TRX over-expression in BEAS-2B cells 24.6 Intracelluar TRX-flag immunostaining 26.7 Apoptosis detection 26.8 ROS detection 27.9 Neutralization of TGF-β1 28.10 Reverse transcription polymerase chain reaction (RT-PCR) 28.11 Quantitative real-time PCR 28.12 TGF-β1 determination of cell lysate 29.13 Western blot assay 29.14 Statistical analysis 30.1 Expression of constructed TRX plasmid in BEAS-2B cells 32.2 Long-term low-dose CS exposure 32.2.1 Cell viability of CS Exposure 32.2.2 TRX over-expressing cells reduced intracellular ROS generation of CS exposure 33.2.3 TRX modulated gene expression of remodeling factors 34.3 Short-term high-dose CS exposure 34.3.1 Cell viability of CS exposure 34.3.2 CS induced ROS generation in BEAS-2B and TRX-TD cells 35.3.3 TRX over-expressing cells resistant to CS-induced apoptosis 35.3.4 CS upregulated TRX expression 36.3.5 CS inhibited TGF-β1 secretion 36.3.6 Gene expression of remodeling factors under CS exposure 37.3.7 TGF-β1 regulated remodeling factors 38.3.8 CS induced MAPK expression 39hapter 4 Discussion 40.1 TRX over-expression reduced CS-induced ROS generation and apoptosis 40.2 Effect on concentration and exposure period of CS 41.3 CS exposure interfered with remodeling factors 41.3.1 TGF-β1 expression correlated with TRX regulation 41.3.2 CS induced EGFR expression 42.3.3 p21 expression following CS exposure 43.3.4 MMP-9 was regulated by TRX expression 44.3.5 TGF-β1 regulated EGFR and MMP-9 expression 45.4 TRX regulated MAPK kinases 46.5 TRX mediated airway remodeling factors and MAPK kinases 47hapter 5 Conclusion 49eferences 80表目錄able 1 Sequences of PCR primers in this study 50igure 1 Framework of study design in the present study 19igure 2 Equipments of cigarette smoke extraction 22igure 3 Protocol of developing a TRX over-expressing cell line 25igure 4 Expression of TRX and TRX-flag in BEAS-2B and TRX-TD cells 51igure 5 Immunofluorescent detection of flag protein in BEAS-2B cells transfected with pTRE2hyg-TRX-3flag plasmid 52igure 6 Cell viability of BEAS-2B and TRX-TD cells in exposure to long-term low-dose CS 53igure 7 ROS generation of BEAS-2B and TRX-TD cells in exposure to long-term low-dose CS. 54igure 8 TRX transcripts following long-term low-dose CS exposure 55igure 9 Gene expression of remodeling factors in response to long-term low-dose CS exposure 56igure 10 Cell viability of BEAS-2B and TRX-TD cells in exposure to short-term high-dose CS 57igure 11 ROS generation of BEAS-2B cells in exposure to short-term high-dose CS 58igure 12 ROS generation of TRX-TD cells in exposure to short-term high-dose CS 59igure 13a Apoptosis and necrosis of BEAS-2B cells in exposure to short-term high-dose CS 60igure 13b Apoptosis of BEAS-2B cells in exposure to short-term high-dose CS 61igure 14a Apoptosis and necrosis of TRX-TD cells in exposure to short-term high-dose CS 62igure 14b Apoptosis of TRX-TD cells in exposure to short-term high-dose CS 63igure 15 Apoptosis in response to H2O2 exposure 64igure 16 TRX expression in response to short-term high-dose CS exposure 65igure 17 TRX expression in response to short-term high-dose CS exposure after TGF-β1 neutralization 66igure 18 CS induced TGF-β1 secretion after short-term high-dose exposure 67igure 19a TGF-β1 expression in response to short-term high-dose CS exposure 68igure 19b EGFR expression in response to short-term high-dose CS exposure 69igure 19c p21 expression in response to short-term high-dose CS exposure 70igure 19d MMP-9 expression in response to short-term high-dose CS exposure 71igure 20a TGF-β1 expression in response to short-term high-dose CS exposure after TGF-β1 neutralization 72igure 20b EGFR expression in response to short-term high-dose CS exposure after TGF-β1 neutralization 73igure 20c p21 expression in response to short-term high-dose CS exposure after TGF-β1 neutralization 74igure 20d MMP-9 expression in response to short-term high-dose CS exposure after TGF-β1 neutralization 75igure 21 Determination of MMP-9 protein using Western assay 76igure 22 Western Blot of BEAS-2B and TRX-TD cells in exposure to 30% CS 77igure 23 Illustration of TRX mediating stress response of cigarette smoke 78application/pdf1021295 bytesapplication/pdfen-US香菸暴露活性氧化物種人類支氣管上皮細胞硫醇氧化還原蛋白呼吸道重塑因子cigarette smokereactive oxidative specieshuman bronchial epithelial cellsthioredoxinremodeling factorsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/181426/1/ntu-98-F91844011-1.pdf