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系統識別號 U0026-1608201215073500
論文名稱(中文) 塵蟎過敏原Der p 2與脂多醣對呼吸道上皮細胞的發炎反應和上皮-間質轉化現象之相互影響
論文名稱(英文) The Interaction of Major Dust Mite Allergen Der p 2 and Lipopolysaccharide in Airway Inflammation and Epithelial-Mesenchymal Transition Changes of Lung epithelial Cells
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 100
學期 2
出版年 101
研究生(中文) 高慧芳
研究生(英文) Hui-Fang Kao
學號 s58921298
學位類別 博士
語文別 英文
論文頁數 102頁
口試委員 召集委員-余俊強
口試委員-謝奇璋
口試委員-葉才明
指導教授-王志堯
口試委員-葉怡玲
口試委員-洪志興
口試委員-林清淵
中文關鍵字 塵蟎  呼吸道上皮細胞  上皮-間質轉化  蜂膠 
英文關鍵字 house dust mites  airway epithelial cells  epithelial-mesenchymal transition  propolis 
學科別分類
中文摘要 氣喘 (bronchial asthma) 是一種異源性呼吸道慢性發炎的疾病,其複雜的病理特徵表現為呼吸道可逆性的呼氣氣流阻塞、發炎、重塑 (remodeling) 及過度反應 (airway hyper-responsiveness, AHR)。超過百分之五十的氣喘患者曾被環境中過敏原所致敏,當患者接觸到空氣中的過敏原例如:塵蟎 (house dust mite)常會發展出偏向第二型輔助性T細胞 (Th2) 的免疫反應。塵蟎過敏原依據基因序列及功能分類目前已有超過20群以上的過敏原被發現。第二群過敏原如Der p 2 和 Der f 2 是塵蟎萃取物中含量高的低分子量非分解酶分子,可以誘發過敏患者體內特異性IgE的產生。流行病學研究指出孩童暴露於多樣性微生物環境中可以防範過敏的發生;而感染和過敏的惡化有關。呼吸道並非無菌,因此來自外界環境或是呼吸道存在的微生物及其成分對過敏反應可能會有影響。最近我們和其他團隊的研究都發現在塵蟎所引起的過敏性發炎反應,特別是塵蟎過敏原合併脂多醣 (lipopolysaccharide, LPS) 所引起的發炎反應中,活化先天性免疫反應扮演很重要的角色。但是我們對於這些來自空氣中的過敏原和脂多醣分子在呼吸道黏膜上彼此間如何相互作用並不了解,因此對這些相互作用是否可能引起發炎反應導致呼吸道上皮細胞的病變進而造成上皮-間質轉化 (epithelial-mesenchymal transition, EMT) 也不清楚。
因此,在這次的研究中,我們利用真菌 Pichia pastoris表現並分離出和IgE具有高度親和力的重組Der p 2 。再將重組Der p 2刺激人類呼吸道上皮細胞株,來探討 (1) 塵蟎過敏原中主要的成分-Der p 2是否可以直接引起呼吸道上皮細胞的發炎反應和上皮-間質轉化現象,以及天然複合物-蜂膠是否能預防transforming growth factor-β1 (TGF-β1) 在呼吸道上皮細胞所誘發的上皮-間質轉化現象。(2) 塵蟎過敏原中主要的成分-Der p 2是否可以放大脂多醣引起的呼吸道發炎反應,並探索的放大反應當中的分子作用機轉。
在我們第一部分的研究顯示,Der p 2不會直接使呼吸道上皮細胞產生上皮-間質轉化現象,所以我們利用很常使用的TGF-β1 誘發A549細胞產生上皮-間質轉化現象當作模組,進行上皮-間質轉化現象的相關實驗。在TGF-β1 誘發A549細胞產生上皮-間質轉化的過程中,包括細胞型態的改變、E-cadherin降低和N-cadherin增加、細胞骨架F-actin的重組、reactive oxygen species (ROS)的產量及細胞移動性等,都會隨著TGF-β1劑量增加變化隨之增強。而預先給予蜂膠,能夠避免TGF-β1作用在A549細胞24小時所誘發的上皮-間質轉化現象,例如維持細胞型態、抑制N-cadherin和ROS的增加、降低F-actin的重組及細胞移動能力;除此之外,蜂膠也能抑制TGF-β1活化下游Smad2及Akt的訊息傳遞路徑及snail 的表現。特別的是蜂膠還可以預防TGF-β1在A549細胞誘發上皮-間質轉化現象時降低的peroxisome proliferator-activated receptor gamma (PPARγ)表現,但是這個預防功能在加入PPARγ的拮抗劑(antagonist)-GW9662之後就失去作用。進一步探討蜂膠成分中重要的活性物質,咖啡酸苯乙酯 (caffeic acid phenethyl ester, CAPE) 和生松素 (pinocembrin) 的功效,發現這兩種物質僅能部分恢復TGF-β1在A549細胞誘發上皮-間質轉化現象所造成的改變,這表示在蜂膠中尚有其它成分參與抑制上皮-間質轉化現象。
第二部分研究著重探討Der p 2在呼吸道上皮細胞以及脂多醣引起的發炎反應中所扮演的病理性角色。將重組Der p 2分別加入A549、H292以及BEAS-2B三種人類呼吸道上皮細胞株,用以評估重組Der p 2在脂多醣引發細胞激素及化學激素產生過程中的功能。結果顯示單獨使用重組Der p 2可使三種細胞株分泌少量的IL-6, IL-8和CCL20。但若重組Der p 2是在脂多醣處理之前加入 (pre-treatment)、之後加入 (post-treatment)、或同時 (co-treatment) 加入刺激細胞株,都比單獨使用脂多醣刺激產生較多的IL-6, IL-8和CCL20。這個增加細胞激素及化學激素分泌的作用主要是經由JNK途徑。相同的實驗結果也可以在重組Der p 2和脂多醣同時 (co-treatment) 嗆入氣管 (intra-tracheal, i.t.) 刺激的小鼠活體得到驗證。重組Der p 2和脂多醣同時刺激相較於單獨重組Der p 2或脂多醣刺激的小鼠,其氣管沖洗液 (bronchoalveolar lavage fluids, BALF) 中含有較高量的IL-6和Thymus activation regulated chemokine (TARC),而且也有較多的浸潤細胞。肺部的組織切片也顯示,重組Der p 2和脂多醣同時刺激的小鼠,其發炎的肺部中有嗜酸性白血球浸潤增多的現象。綜合以上的實驗結果,我們推測重組Der p 2過敏原可以參與並增強脂多醣引起的呼吸道發炎反應,並將其導向類似氣喘性發炎反應-嗜酸性白血球浸潤及以第二型輔助性T細胞 (Th2) 化學激素分泌為主的發炎反應。
蜂膠是種廣泛使用的民俗藥物具有潛在性調節免疫反應的藥物效用。天然蜂膠萃取物可經由多組訊息傳遞途徑抑制TGF-β1誘發A549人類肺泡上皮細胞株產生上皮-間質轉化現象,未來在臨床上可能可以應用於預防或治療與上皮-間質轉化現象有關的肺部纖維化疾病以及慢性氣喘引起的呼吸道重塑病變。
而Der p 2可參與和增強脂多醣誘導的發炎反應,本研究再次確認脂多醣在塵蟎引起的過敏反應扮演一個很重要的角色,提醒我們在處理患者過敏反應時不要忽略脂多醣可能產生的影響,適時控制脂多醣或許有助於阻止過敏反應的惡化。未來還會更多脂多醣對呼吸道作用的研究持續進行,或許這是阻止氣喘慢性發炎進行的新方向。
英文摘要 Bronchial asthma is a heterogeneous inflammatory airway disease; it develops a complex pathology that is characterized by reversible expiratory airflow limitation and airway inflammation accompanying with airway remodeling and airway hyper- responsiveness (AHR). Over 50% of individuals who suffered with asthma have been sensitized with environmental allergens such as house dust mites (HDM) and developed a Th2-biased immune response. More than 20 HDM allergen groups have been defined based on sequence and functional homologies. Belonging to group 2 allergens, Der p 2 and Der f 2 are low molecular weight protein without proteolytic activity that can induce specific IgE antibodies production in allergic patients. Epidemiological studies showed that children exposed to an environment with rich microbes may be protected from allergic asthma; however, infections are likely to be connected with asthma exacerbation. The airway is not sterile; therefore, the microbes from the external environment and the microbes residing in the airway may have great influences on asthma development. Recently, others’ studies and ours have demonstrated that activation of the innate immunity also plays a critical role in HDM-induced allergic inflammation, particularly in the combination with environmental endotoxins. The molecular interaction between aeroallergens and LPS in the mucosal membranes of airway is still not clear, and whether this interaction will cause the pathological effect that lead to epithelial-mesenchymal transition (EMT) in the airway epithelial cells is also unknown.
Therefore, in this study, we expressed, isolated, and applied recombinant Der p 2 (rDer p 2) with high IgE binding activity from Pichia pastoris to human airway epithelial cell lines to investigate (1) whether the major HDM allergen, Der p 2, could directly induce inflammation and EMT in airway epithelial cells, and if so whether the natural product of propolis could have preventive effects in the TGF-β1-induced EMT in airway epithelial cells; (2) whether the major HDM allergen, Der p2, could augment LPS-induced airway inflammation, and if so, explored the possible molecular mechanisms involved in this augmentation effect.
In the first part of our research, the results showed that stimulation of rDer p 2 alone could not induce EMT in airway epithelial cells. Therefore, we used a well-established model, TGF-β1-induced-EMT in A549 cells, to perform our experiments on EMT. Experimental results showed progressive cell morphological change, decreased E-cadherin production, increased N-cadherin production, intracellular F-actin rearrangement, increased ROS production, and increased cell motility with an increase in the concentrations of TGF-1 in A549 cells. Pretreated with propolis and then treated with TGF-1 for 24 h regained epithelial cells morphology and decreased the production of N-cadherin and ROS and decreased cell motility. Propolis prevented the effect of TGF-1-induced Smad2 and Akt activation pathways and Snail expression. Moreover, propolis pretreatment may prevented the TGF-1-induced down-regulation of nuclear hormone receptor and peroxisome proliferator-activated receptor gamma (PPARγ) protein in A549 cells, whose effect was blocked by adding PPARγ antagonist, GW9662. Two active components of propolis, caffeic acid phenethyl ester (CAPE) and pinocembrin (PIN) only had partial effects on TGF-β1-induced EMT in A549 cells. The results of this study suggest that other unidentified components of propolis may be involved in the inhibitory effect on TGF-1-induced EMT in A549 cells.
In the follow-up research, the aim was set to explore the pathological roles of Der p 2 in airway epithelia cells, and its effect in LPS-induced airway inflammation. We used rDer p 2 to treat human airway epithelial cell lines, BEAS-2B, A549, or H292 to evaluate the effect of Der p 2 on LPS-induced cytokines or chemokines production. rDer p 2 alone could not induce high levels of IL-6, IL-8 or CCL20. We then added rDer p 2 with LPS. In any case (pre-treatment, co-treatment, and post-treatment), the production levels of IL-6, IL-8 or CCL-20 were higher than those by adding LPS only. This enhancement of inflammatory cytokines production was through JNK signal pathway. These results were further confirmed by using in vivo experiment, which administrated intra-tracheally rDer p 2 and LPS simultaneously into the lungs of mice. These mice produced higher IL-6 and TARC, and the amount of infiltrated cells in their bronchoalveolar lavage fluids (BALF) was larger than mice stimulated with rDer p 2 or LPS alone. In conclusion, our results suggest that recombinant Der p 2 allergen can participate and enhance LPS-induced airway inflammation into eosinophil infiltration and Th2 chemokine predominant, asthma-like inflammation.
Propolis is a popular folk medicine with potential pharmacological actions related to immunological reactions. The results of this study suggest that natural propolis extracts may prevent TGF-β1-induced EMT in immortalized type II AECs via multiple inhibitory pathways, which may be clinically applied in the prevention and/or treatment for EMT-related fibrotic diseases as well as airway remodeling in chronic asthma.
Der p 2 allergen can participate and enhance LPS-induced airway inflammation. This study also suggested that LPS play an import role in allergy; the phenomenon that Der p 2 and LPS may work together to deteriorate inflammatory reactions reminds us the potential affects from LPS to allergy development cannot be ignored. Appropriate and in-time control over LPS may keep allergy from exacerbation. In the future, further investigations about the effect of LPS in the airway will be continued. There may be novel targets for stopping the progression from chronic inflammation of asthma.
論文目次 Contents
Chinese Abstract I
English Abstract IV
Contents VII
List of Tables XI
List of Figures XII
I. Introductions 1
1. Heterogeneity of asthma 1
2. Pathologic characters of allergic asthma 2
3. Allergic asthma and airway epithelial cells (AECs) 3
4. Allergic asthma and airway remodeling 3
5. Epithelium repair and epithelial-mesenchymal transition (EMT) 4
6. The role of TGF-1 on EMT in asthma 5
7. Propolis and its therapeutic effect 5
8. Allergens of house dust mite 6
9. Structural similarity between Der p 2, Der f 2, and MD-2 7
10. Relation of LPS and MD-2 in TLR4 signaling 8
II. Specific Aims 11
1. Specific aim A: to explore the roles of HDM allergen and propolis in the EMT of airway epithelial cells 12
(1) To explore the roles of major HDM allergen Der p 2 in the EMT of airway epithelial cells 12
(2) To explore the effect of propolis in preventing EMT in airway epithelial cells 13
2. Specific aim B: to explore the role of allergens in promoting inflammation in airway epithelial cells 14
(1) To verify whether the major HDM allergen Der p 2 can directly induce inflammation in airway epithelial cells 15
(2) To verify whether the major HDM allergen Der p 2 can augment LPS-induced immune response in airway epithelial cells 15
(3) To explore the possible molecular mechanism involved in Der p2 augment LPS-induced immune response 16
III. Materials and Methods 17
1. Reagents 17
2. Equipment 18
3. Preparation of Der p extract 18
4. Expression and purification of recombinant Der p 2 in Pichia pastoris 19
5. Preparation of ethanolic extracts of red propolis and HPLC analysis 20
6. Cell culture 20
7. Stimulation of cells 21
8. Immunofluorescence staining 22
9. Intracellular reactive oxygen species (ROS) assay 23
10. F-actin staining (phalloidin staining) 23
11. Migration assay 23
12. Western blot 24
13. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis 25
14. Real-time PCR 27
15. ELISA of cytokine level 27
16. Mice 28
17. Statistical analysis 29
IV. Results 30
1. PART A: to explore the roles of HDM allergen and propolis in the EMT of airway epithelial cells 30
(1) Expression and purification of rDer p 2 in Pichia pastoris 30
(2) rDer p 2 allergen cannot induce EMT in airway epithelial cells 31
(3) TGF-β1 induces EMT in A549 human lung epithelial cells 32
(4) TGF-1 induces ROS production, stress fiber formation and cellular migration during EMT of A549 cells 33
(5) Propolis inhibits TGF-1-induced EMT in A549 cells 34
(6) Propolis inhibits TGF-β1-induced ROS production, stress fiber formation and cellular migration during EMT of A549 cells 34
(7) Propolis inhibits TGF-β1-induced Smad2, Akt, and Snail phosphorylation during EMT of A549 cells 35
(8) CAPE and PIN partially inhibits TGF-β1-induced EMT in A549 cells 36
(9) Propolis inhibits TGF-β1-induced PPARγ down regulation during EMT of A549 cells 37
(10)Summary 38
2. PART B: to explore the role of allergens in promoting inflammation in airway epithelial cells 40
(1) Pretreatment with rDer p 2 increases mRNA production of pro-inflammatory cytokines in LPS-stimulated epithelial cells 40
(2) Treatment with rDer p 2 increases pro-inflammatory cytokines production in LPS-stimulated airway epithelial cells 41
(3) rDer p 2 can augment LPS-induced inflammatory reaction by itself 42
(4) rDer p 2 augments LPS-induced inflammatory responses in airway epithelial cells through JNK phosphorylation 43
(5) Combinational treatment with rDer p 2 and LPS augments airway inflammation in mice 45
(6) Summary 45
V. Discussion 47
1.Propolis inhibits TGF-β1-induced EMT in human alveolar epithelial cells via PPARγ..................................................................................................47
2.The relation between PPARγ and fibrosis…… 49
3.The relation between propolis and PPARγ during TGF-β1-induced EMT 50
4 The augmentation effect of mite allergen, Der p 2, on LPS-induced airway inflammation 52
5.The role of Der p 2 in LPS-induced TLR4 signaling 52
6.The inflammatory reaction among pre-, co-, and post-treatment with Der p 2 in LPS-stimulated cells 55
7. Future work 56
VI. Supplementary Figures 90
VII. References 91

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