臺灣博碩士論文加值系統

過敏性氣喘是一種慢性的支氣管發炎疾病，一般認為，具有過敏體質的人對於吸入特定的過敏原會產生不正常的免疫反應。過去的研究發現，第二型T輔助細胞(Th2細胞)被認為是主要的調控者，Th2細胞所產生的細胞激素IL-4、IL-5、IL-13在引起氣喘的病發過程中扮演重要的角色，這些Th2細胞激素會導致肥大細胞和嗜酸性白血球的分化與活化，並且吸引這些發炎細胞到支氣管和肺臟，這些活化的發炎細胞會分泌大量的發炎調控因子進而引起支氣管阻塞、呼吸道過度反應和發炎，而導致氣喘。最近，一個新的four-helix bundle細胞激素IL-31被發現，主要由活化的T細胞所分泌；其中，特別是Th2細胞的表現量比Th1細胞來得多。在以皮下注射IL-31蛋白質的小鼠中發現，過度地表現IL-31會出現類似皮膚炎的病徵，而臨床上，幾乎80％患有異位性皮膚炎的小孩將來都可能發展成過敏性鼻炎和氣喘。IL-31的訊息主要是透過IL-31 receptor A (IL-31 RA)和oncostatin M receptor (OSMR)所形成的heterodimeric receptor所傳遞。不過，目前對IL-31詳細的生物功能還不是十分清楚；但除了在上皮細胞和角質細胞上固定表現有IL-31 receptor，利用ovalbumin (OVA)致敏的小鼠當中，在肺臟、肺臟上皮細胞和肺泡沖洗液細胞中也有較高的IL-31 RA mRNA的表現。
因此，我們想評估是否IL-31在氣喘發病的過程中扮演重要的角色。首先，我們從活化的T細胞中clone到IL-31 cDNA並且建構到pcDNA3.1真核表現載體中，利用肌肉注射方式分別在第0天和第7天，將pcDNA3.1/mIL-31打入到正常的小鼠中，結果發現，在施打IL-31的老鼠的肺泡沖洗液中有大量T細胞浸潤的現象。我們進一步利用ovalbumin作為過敏原的小鼠氣喘模式，探討IL-31在氣喘過程中可能扮演的角色，同樣利用肌肉注射的方式分別在致敏流程第13天和第20天，將pcDNA3.1/mIL-31打入到OVA致敏的小鼠中。小鼠犧牲後，抽取肺臟與肺泡沖洗液細胞的mRNA，利用RT-PCR分析IL-31和receptors在OVA致敏小鼠中的表現量，以ELISA方式定量血清中OVA專一性抗體和OVA刺激脾臟細胞所分泌的Th2細胞激素。結果發現，IL-31和OSMR在對照組和OVA致敏小鼠中的表現量沒有明顯的差異，而IL-31 RA在OVA致敏小鼠的肺泡沖洗液細胞和肺臟中有明顯較高的表現量。血清抗體方面，OVA致敏小鼠與對照組比較發現，OVA專一性抗體的量沒有顯著增加的情形，而OVA刺激脾臟細胞培養部分，在施打IL-31的OVA致敏小鼠中，Th2細胞激素的表現量明顯地比對照組來得高。根據這些結果，我們目前假設在OVA致敏小鼠中，IL-31主要是透過高表現量的IL-31 RA傳遞訊息進而加重氣喘的發病情形。

Asthma is a complex pulmonary inflammatory disease. Generally, it is believed that the susceptible individuals have an abnormal immune response to inhaled allergens. According to the previous studies, Th2 cells and their cytokines, IL-4, IL-5, and IL-13 play a pivotal role in the pathogenesis of asthma. These Th2 cytokines cause the differentiation, recruitment, and activation of the mast cells and eosinophils in the airway mucosa. Activation of these cells releases a large number of proinflammatory mediators which induce bronchial obstruction, airway hyper-responsiveness, and airway inflammation. Recently, a novel four-helix bundle cytokine, IL-31, has been identified. IL-31 is expressed mainly by activated T cells, produced particularly in larger amount in Th2 than Th1 cells. IL-31 induces similar symptoms of dermatitis in mice. Clinically, nearly 80% children with atopic dermatitis develop allergic rhinitis or asthma. IL-31 signals via the heterodimeric receptor complex composed of IL-31 RA and OSMR. However, the exact biological functions of IL-31 aren’t fully understood. Besides in epithelial cells and keratinocytes, IL-31 receptors are up-regulated in lung epithelium and bronchoalveolar lavage cells from an animal model of OVA-challenged airway hyper-responsiveness. Thus, we would like to evaluate whether IL-31 plays a role in the pathogenesis of asthma. We first cloned IL-31 cDNA from activated splenocytes and constructed into the eukaryotic pcDNA3.1/mIL-31 expression vector. Normal mice were injected intramuscularly by pcDNA3.1/mIL-31 at days 0 and 7. Increased numbers of lymphocytes in BALF were significantly detected from IL-31 administrated mice. We further used OVA as an allergen to induce an asthmatic animal model; the sensitized mice were injected intramuscularly by pcDNA3.1/mIL-31 at days 13 and 20. The expression of IL-31 and receptor genes in BALF cells and lung tissues of OVA-sensitized mice was analyzed by RT-PCR. The OVA-specific antibody from serum and Th2 cytokines from OVA-treated splenocytes were quantified by ELISA. Nevertheless, the levels of IL-31 and OSMR in lung tissues between normal mice and OVA-sensitized mice were not different. IL-31 RA in the lung tissues and BALF cells of OVA-sensitized mice expressed significantly higher levels than normal mice. The titers of OVA-specific antibody between normal and OVA-sensitized mice were not different. Elevated Th2 cytokines were expressed in splenocytes of pcDNA3.1/mIL-31 injected OVA-sensitized mice. Our data suggest that IL-31 may enhance the pathogenesis of asthma through higher expression of IL-31 RA in OVA-sensitized mice

Contents

Acknowledgements…………………………………………………...i
Abstract (Chinese)…...………………………………………………ii
Abstract (English)…………………………………………………...iv
Contents………………………………………………………………..vi


 CHAPTER I Introduction…………………………………...1
1.1 T helper cells and cytokines in asthma……………………….4
1.2 Interleukin-31 and receptors………………………………….7

 CHAPTER II Materials and Methods…………………..11
2.1 Gene cloning…………………………………………………..11
2.2 Animal study………………………………………………….18

 CHAPTER III Results...…………………………………….25
3.1 The cloning of mouse IL-31 gene.............................................25
3.2 The percentage of lymphocyte in BALF of normal mice
that injected intramuscularly with pcDAN3.1/mIL-31 plasmid DNA………………………………………………….25
3.3 The airway hyper-responsiveness of OVA-sensitized mice...26
3.4 The percentage of eosinophils in BALF……………………..27
3.5 OVA-specific antibody in mice serum………………………27
3.6\ Th2 cytokines in culture supernatant of OVA-sensitized mice……………………………………..……………………28
3.7 The expression of mIL-31, mIL-31 RA and OSMR mRNA in whole lung tissues and BALF cells of OVA-sensitized mice..29
3.8 The expression of mIL-31 recombinant protein in BL21 (DE3) cells with pET29a(+)/mIL-31………………………………...30

 Chapter IV Discussion............................................................32

 Chapter V Futurework……………………...........................36

 References..………………………………………………………37

 Tables and Figures………………………………………..……45

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