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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 莊雅惠 | |
| dc.contributor.author | Pin-Yi Lin | en |
| dc.contributor.author | 林品儀 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:25:35Z | - |
| dc.date.available | 2022-12-31 | |
| dc.date.copyright | 2012-09-18 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-31 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65111 | - |
| dc.description.abstract | 過敏性呼吸道疾病是一種慢性發炎疾病，造成的原因為Th2免疫反應失衡。其機制是由於病人呼吸道對特定的外來物質(過敏原)產生過敏反應，導致過敏原專一性Th2細胞的活化、Th2相關的細胞激素IL-4、IL-5和IL-13大量產生、血管擴張、平滑肌收縮、黏液以及抗體IgE的產生，造成呼吸道發炎。先前的文獻指出，在過敏性鼻炎動物模式中，IL-21藉由降低B細胞產生IgE的能力減緩過敏性鼻炎的症狀。但IL-21是否影響Th2細胞則尚未明瞭。在此實驗中，我們藉體內與體外試驗探討IL-21是否調控Th2細胞的分化與Th2免疫反應。
首先，我們探討IL-21是否影響Th2細胞分化。將DO11.10小鼠的CD4 T細胞以Th2條件培養時同時加入不同濃度之rmIL-21後分析培養細胞分泌的Th2相關細胞激素，結果顯示，加入IL-21培養的細胞其分泌Th2相關細胞激素的能力降低，其中以IL-4下降最為明顯，且Th2細胞激素的下降並非IL-21造成細胞死亡所致。再者，利用在小鼠致敏前給予Ad-mIL-21觀察其於Th2-derived過敏性動物模式對Th2細胞分化的影響。與施打Ad-mock的小鼠相比，接受Ad-mIL-21的小鼠支氣管肺泡沖洗液中IL-4、IL-5與IL-13濃度亦顯著下降。 接著，我們探究IL-21是否調控Th2免疫反應。以不同濃度rmIL-21刺激小鼠Th2條件培養後的細胞，發現細胞分泌的Th2相關細胞激素(IL-4、IL-5與IL-13)降低。再者，利用給予已致敏小鼠Ad-mIL-21後，支氣管肺泡沖洗液中IL-4濃度顯著下降。進一步分析發現，IL-4濃度的下降並非IL-21造成CD4 T細胞死亡所致而可能為藉由降低GATA3的表現而抑制細胞分泌IL-4。最後Th2相關細胞激素受到IL-21影響而降低的情形同樣能在以不同濃度rhIL-21刺激過敏性疾病病人分離出之周邊血單核球細胞的實驗中觀察到。 綜合以上實驗，IL-21能抑制Th2細胞的分化，並抑制Th2細胞的功能。其可能的機制為IL-21藉由負向調控GATA3與降低IL-4於肺臟中之表現量抑制過度的Th2免疫反應，並非IL-21造成Th2細胞死亡導致Th2相關細胞激素減少。 | zh_TW |
| dc.description.abstract | Allergic airway disease is a chronic inflammation, which is regarded as Th2 weighted imbalance. After allergens or parasitic helminthes stimulation, the phenomena of eosinophil increase, mast cell activation, immunoglobulin E (IgE) antibody production and cytokines such as interleukin (IL)–4, IL-5, and IL-13 secreted by Th2 cells are contributed to allergic disease. Previous study suggests that IL-21 administration into the nostril alleviates murine allergic rhinitis by means of reducing the production of IgE. However, whether IL-21 affects Th2 cells is still unknown. In this study, we investigated the role of IL-21 in the regulation of Th2 cell differentiation and Th2 immune responses in vitro and in vivo.
First, we explored whether IL-21 affects the Th2 cell differentiation. The secretion of IL-4 from DO11.10 CD4 T cells which were cultured with different concentration of IL-21 under Th2 polarizing condition was decreased. In addition, we confirmed that the decreased level of IL-4 was not caused by IL-21-inducing apoptosis of cells. Furthermore, we also observed the significantly decreased level of Th2-associated cytokines in the BALF of OVA/Alumimmunized mice which were received Ad-mIL-21 before OVA immunization. Besides, the administration of Ad-mIL-21 reduced IL-4 production in the BALF of OVA/Alum immunized mice. Furthermore, IL-21 administration decreased the expression of transcription factor GATA3 of T cells. In conclusion, our results demonstrated that IL-21 decreased the Th2 cell differentiation and Th2 immune response. Moreover, the effects of IL-21 in the down regulation of Th2 cells were resulted in the down-regulation of GATA3 but not the apoptosis of Th2-producing cells. | en |
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| dc.description.tableofcontents | 致謝 i
中文摘要 ii Abstract iv 縮寫對照表 vi 第一章 研究背景 1 1.1 呼吸道發炎反應(airway inflammatory response) 1 1.1.1 樹突細胞(dendritic cells，DCs) 1 1.1.2 呼吸道上皮細胞(airway epithelial cells) 1 1.1.3 肥大細胞(mast cells) 2 1.1.4 嗜酸性球(eosinophils) 2 1.1.5 B淋巴球(B lymphocytes) 3 1.1.6 T淋巴球(T lymphocytes) 3 1.2 過敏機轉(Allergic mechanism) 4 1.2.1 CD4+ T細胞免疫反應 4 1.2.2 第二型輔助型T細胞免疫反應之起源(The orgin of Th2 immune response) 4 1.2.3 致敏(sensitization)與記憶(memory) 5 1.2.4 挑戰期(Challenge phase) 5 1.2.5 晚期(Late phase response) 6 1.3 介白素-21(Interleukin-21) 6 1.3.1 介白素-21與介白素-2家族 6 1.3.2 介白素-21於先天免疫與後天免疫之調控 6 1.3.3 介白素-21於B細胞功能上的調控 7 1.3.4 介白素-21與Th1免疫反應 7 1.3.5 介白素-21與Th17免疫反應 8 1.3.6 介白素-21與調控型T細胞(regulatory T cells)之關係 8 1.3.7 介白素-21與Th2免疫反應(過敏反應) 8 1.4 基因療法(Gene therapy) 9 1.4.1 腺病毒載體(Adenovirus vector) 9 1.4.2 腺病毒載體與細胞性免疫反應 10 1.4.3 腺病毒載體與嗜中性球之浸潤 10 1.5 研究目的 10 第二章 實驗材料與方法 11 2.1 探討rmIL-21於體外試驗(in vitro)中對Th2細胞之影響 11 2.1.1 脾臟細胞之純化 11 2.1.2 CD4 T細胞分離純化 11 2.1.3 Th2-derived培養環境 12 2.1.4 流式細胞儀(flow cytometry) 分析細胞表面抗原與細胞內細胞激素 12 2.1.5 細胞死亡之觀察 13 2.2 細胞RNA之萃取 13 2.3 利用RNA反轉錄成cDNA之過程 14 2.4 以腺病毒載體(Adenovirus Vector)系統表現小鼠IL-21 14 2.4.1 小鼠IL-21基因片段與pShuttle-Hpgk．B1質體製備 14 2.4.1.1 聚合酶鏈鎖反應(Polymerase chain reaction；PCR) 15 2.4.1.2 小鼠基因片段與質體接合之製備 15 2.4.1.3 質體轉殖(transformation)入E.coli DH5α 15 2.4.1.4 質體DNA之萃取 16 2.4.1.5 質體轉染(transfection)至COS-1細胞 16 2.4.2 pShuttle-Hpgk．B1質體與pAdEasy-1質體進行同源重組(homologous recombination) 16 2.4.2.1 篩選成功重組之pAdEasy-1- pShuttle-Hpgk．B1 16 2.4.3 將重組之質體轉染至AD-293細胞 17 2.5 重組腺病毒增量(amplify)與置備 17 2.5.1 病毒感染(infection)與培養 17 2.5.2 病毒增量 17 2.5.3 病毒純化 18 2.5.4 病毒定量 18 2.6 實驗用小鼠 18 2.7 Th2-mediated過敏反應動物模式 19 2.7.1 血清樣品之收集 19 2.7.2 以Enzyme linked immunosorbent assay (ELISA)偵測血清OVA-specific IgE 20 2.7.3 小鼠支氣管肺泡沖洗液(bronchoalveolar lavage fluid;BALF)之收集與處理 20 2.7.4 小鼠支氣管肺泡沖洗液之細胞離心與染色 21 2.7.5 小鼠支氣管肺泡沖洗液之細胞分類 21 2.7.6 小鼠支氣管肺泡沖洗液淋巴球細胞之組成 21 2.7.8 ELISA偵測細胞激素(IL-4、IL-5、IL-10、IL-13、IL-17、IL-21、IFN-γ與TGF-β) 22 2.7.9 以即時定量反轉錄聚合酶連鎖反應偵測GATA3 22 2.8 探討rhIL-21刺激人類PBMC之情形 23 2.8.1 病人檢體 23 2.8.2 由全血分離 PBMC (Peripheral blood mononuclear cell) 23 2.8.3 以PBMC體外刺激(in vitro stimulation) 24 2.8.4 以Enzyme linked immunosorbent assay(ELISA) 測量上清液細胞激素之濃度 24 2.9 繪圖與統計分析 24 第三章 結果 25 3.1 相較於Th0細胞，經Th2條件培養的細胞能表現較高的IL-4 / IFN-γ比例 25 3.2 IL-21抑制Th2細胞的分化而促進Th17細胞的生成 25 3.3 IL-21不會造成預分化成Th2的細胞走向死亡 26 3.4 製備攜帶小鼠IL-21的重組腺病毒 26 3.5 Ad-mIL-21能於小鼠體內表現IL-21 27 3.6 先接受Ad-mIL-21再以OVA致敏之小鼠其肺臟中IL-4、IL-5、IL-13與IL-17的產量顯著下降 27 3.6.1 先接受Ad-mIL-21再以OVA致敏之小鼠血清中 OVA 特異性 IgE未受影響 27 3.6.2 先接受Ad-mIL-21再以OVA致敏之小鼠肺臟內嗜中性球浸潤的比例上升 28 3.6.3 先接受Ad-mIL-21再以OVA致敏之小鼠肺臟內細胞激素IL-4、IL-5、IL-13與IL-17之產量達到顯著下降 28 3.7 IL-21直接地抑制Th2條件培養的細胞分泌Th2相關細胞激素的能力 29 3.8 IL-21不會造成Th2條件培養的細胞走向死亡 30 3.9 接受Ad-mIL-21的OVA致敏小鼠雖有發炎反應但CD4 T細胞分泌IL-4的能力下降 30 3.9.1 以OVA致敏之小鼠於接受Ad-mIL-21後，血清中 OVA 特異性 IgE未受影響 30 3.9.2 OVA致敏之小鼠於接受Ad-mIL-21後，並無影響肺臟內浸潤細胞數與各個浸潤細胞之比例 30 3.9.3 OVA致敏之小鼠於接受Ad-mIL-21後，其肺臟內IL-4的濃度下降 31 3.10 人類周邊血單核球細胞於IL-21刺激後分泌IL-5的能力下降而IL-17的濃度升高 32 第四章 討論 33 圖 38 參考文獻 62 附錄 73 | |
| dc.language.iso | zh-TW | |
| dc.subject | IL-21 | zh_TW |
| dc.subject | IL-4 | zh_TW |
| dc.subject | Th2細胞 | zh_TW |
| dc.subject | 過敏性疾病 | zh_TW |
| dc.subject | 支氣管肺泡沖洗液 | zh_TW |
| dc.subject | GATA3 | zh_TW |
| dc.subject | broncho-alveolar lavage fluid (BALF) | en |
| dc.subject | Th2 cells | en |
| dc.subject | allergic disease | en |
| dc.subject | IL-21 | en |
| dc.subject | IL-4 | en |
| dc.subject | GATA3 | en |
| dc.title | IL-21於調控Th2細胞分化與Th2免疫反應之角色 | zh_TW |
| dc.title | The roles of IL-21 in the regulation of Th2 cell differentiation and Th2 immune responses | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊雅倩,李岳倫,張純榮 | |
| dc.subject.keyword | IL-21,IL-4,Th2細胞,過敏性疾病,支氣管肺泡沖洗液,GATA3, | zh_TW |
| dc.subject.keyword | IL-21,IL-4,Th2 cells,allergic disease,broncho-alveolar lavage fluid (BALF),GATA3, | en |
| dc.relation.page | 77 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-01 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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