臺灣博碩士論文加值系統

過敏是免疫介導的疾病，主要由對過敏原IgE依賴性免疫反應所引起，而氣喘就是一種過敏性的疾病，它被定義為過敏原介導的呼吸道炎症性疾病，在嚴重患者中常會出現咳嗽、胸悶、喘不過氣等症狀，目前用來治療氣喘的藥物只能用來緩解，並常伴有嚴重的副作用。許多文獻指出綠藻的硫酸多醣具有多種生理活性，而胚芽乳酸桿菌 (Lactobacillus plantarum) 具有降低腸胃道疾病風險、免疫調節、抗過敏多種功效，多篇研究指出合生元不僅用於提高添加到食品或飼料中的有益微生物的存活率，還用於刺激胃腸道中存在的特定天然菌株的增殖。本研究主旨為探討石蓴多醣與胚芽乳酸桿菌聯合給予，是否改善呼吸道過敏，細胞實驗以化合物48/80誘導P815肥大細胞脫顆粒實驗結果顯示共同給予胚芽乳酸桿菌以及石蓴會隨著劑量依賴性抑制脫顆粒的現象，動物實驗以OVA誘導的氣喘小鼠模型，分為六個組別 (控制組、藥物控制組、OVA誘導組、胚芽乳酸桿菌組、石蓴多醣組、混合組)，在肺臟以及脾臟組織重量分析上，跟OVA組別相比給予石蓴、胚芽乳酸桿菌及兩者共同給予的組別重量顯著降低，表示當給予樣品後降低了肺部發炎情形並且有效調控免疫反應，在血液分析中發現給予樣品後白血球、淋巴球、中間型細胞皆顯著下降，且血紅蛋白出現上升情形，表示透過給予胚芽乳酸桿菌及石蓴能夠有效減緩OVA引起的發炎反應並提高血液中氧氣的攜帶量來改善氣喘，且流式細胞儀分析結果顯示，輔助型T細胞及毒殺型T細胞皆有顯著升高，並透過酵素結合免疫吸附法 (ELISA) 觀察小鼠血液及支氣管肺部沖提液中IgE、IgG2a、IgG1含量，發現給予樣品後IgE、IgG1皆有出現下降的情形，而IgG2a則有升高的趨勢；後續再透過 qPCR 觀察其使IL-4、IL-5、IL-13、IL-17下降，並使 IFN-γ 、IL-10上升，並且透過短鏈脂肪酸分析可以看到其乙酸、丙酸及丁酸濃度皆有顯著增加的趨勢，其中在混合組效果最為明顯，綜合上述結果可以看到不管是單獨給予石蓴及胚芽乳酸桿菌，或是將兩者混合給予後皆能抑制氣喘，且透過聯合給予的方式使短鏈脂肪酸增加來達到減緩過敏性氣喘的效果。

Allergies are immune-mediated diseases primarily caused by IgE-dependent immune responses to allergens. Asthma is a type of allergic disease defined as an allergen-mediated respiratory inflammatory condition. In severe cases, symptoms such as coughing, chest tightness, and difficulty breathing can occur. Current medications for asthma provide only symptomatic relief and often come with serious side effects. Numerous studies have indicated that green algae polysaccharides possess various physiological activities. Additionally, Lactobacillus plantarum, a strain of lactobacillus found in germ buds, offers multiple benefits including reducing the risk of gastrointestinal disorders, immune regulation, and anti-allergic effects. Several research papers have suggested that synbiotics are used not only to enhance the survival rate of beneficial microorganisms added to food or feed but also to stimulate the proliferation of specific natural strains in the gastrointestinal tract. The focus of this study is to investigate the combined administration of green algae polysaccharides and Lactobacillus plantarum to improve respiratory allergies. In cell experiments, the compound 48/80 induced P815 mast cell degranulation, and the results showed that mixture of Lactobacillus plantarum and Ulva resulted in dose-dependent inhibition of degranulation. In animal experiments using an OVA-induced asthma mouse model, six groups were formed: control group, drug control group, OVA-induced group, Lactobacillus plantarum group, Ulvan group, and mixture group. Analysis of lung and spleen tissue weights revealed significant reductions in the weight of the Ulva, Lactobacillus plantarum, and mixture groups compared to the OVA group. This indicates a decrease in lung inflammation and effective immune regulation after sample administration. Blood analysis showed significant decreases in white blood cells, lymphocytes, and neutrophils, along with an increase in hemoglobin levels, suggesting that administration of Lactobacillus plantarum and Ulva can effectively alleviate OVA-induced inflammation and improve asthma by enhancing oxygen-carrying capacity in the blood. Flow cytometry analysis indicated significant increases in helper T cells and cytotoxic T cells. Enzyme-linked immunosorbent assay (ELISA) observations of IgE, IgG2a, and IgG1 levels in mouse blood and bronchoalveolar lavage fluid revealed decreases in IgE and IgG1, with an increasing trend in IgG2a after sample administration. Subsequent qPCR analysis demonstrated decreases in Th2, and Th17, and increases in Th1 and Treg. Short-chain fatty acid analysis showed significant increases in acetic acid, propionic acid, and butyric acid concentrations, with the Mixture group showing the most pronounced effect. In conclusion, whether administered separately or in combination, Ulva and Lactobacillus plantarum effectively inhibit asthma. The combined administration approach enhances short-chain fatty acid production, contributing to the alleviation of allergic asthma.

摘要 I
Abstract I
圖目錄 VI
前言 1
第一章、 文獻回顧 2
一、 氣喘 (ASTHMA) 2
1. 氣喘之介紹 2
2. 氣喘之流行病學 2
3. 氣喘之類型 3
3-1. 外因性氣喘 (Extrinsic asthma) 3
3-2. 內因性氣喘 (Intrinsic asthma) 3
4. 氣喘之臨床病徵 3
4-1. 氣道高反應性 (Airway hyperresponsiveness, AHR) 4
4-2. 氣道重塑 (Airway remodeling) 4
5. 過敏反應 (Hypersensitivity reactions) 4
5-1. 第一型過敏反應 (Immediate hypersensitivity reaction) 5
5-2. 第二型過敏反應 (Cytotoxic hypersensitivity reaction) 5
5-3. 第三型過敏反應 (Immune complex-mediated hypersensitivity) 5
5-4. 第四型過敏反應 (cell- mediated hypersensitivity reaction ) 6
6. 已知氣喘機制 6
6-1. Th1 與 Th2 細胞 6
6-2. Th17 與Treg 細胞 6
7. 氣喘相關研究之小鼠動物模式 7
7-1. 卵清蛋白 (Ovalbumin, OVA) 7
8. 目前對氣喘上的治療及因應對策 7
二、 肥大細胞 8
1. 脫顆粒作用 (Degranulation) 8
2. 化合物 48/80 (Compound 48/80) 8
三、 益生菌 (PROBIOTICS) 9
1. 益生菌之介紹 9
1-1. 益生菌的功效及作用機制 9
1-1-1. 增強上皮屏障 9
1-1-2. 增加對腸黏膜的黏附 9
1-1-3. 病原微生物的競爭排斥 10
1-1-4. 抗菌物質的生產 10
1-1-5. 免疫調節 10
2. 胚芽乳酸桿菌 (Lactobacillus plantarum) 11
四、 益生元 (PREBIOTICS) 11
1. 益生元介紹 11
1-1. 益生元功效及機制 12
五、 合生元 (SYBIOTICS) 12
1. 合生元介紹 12
1-1. 合生元功效及機制 12
六、 海藻 (SEAWEED) 14
1. 海藻簡介 14
2. 台灣海藻分布 14
3. 綠藻植物門 (Chlorophyta) 14
4. 石蓴 (Ulva lactuca) 14
5. 石蓴多醣 (Ulvan) 15
第二章、 實驗設計流程 16
一、 樣品製備 16
二、 石蓴特性分析 16
三、 細胞實驗 17
四、 動物實驗 17
1. 日程與分組 17
1-1. 動物實驗日程 17
2. 臟器與分析 17
第三章、 實驗材料與方法 19
一、 實驗材料 19
1. 樣品原料 19
2. 化學性分析試驗 19
3. 細胞株 19
4. 細胞培養之培養基 19
5. 細胞存活率試劑 20
6. P815 脫顆粒誘導試劑 20
7. 一氧化氮試驗 20
8. 實驗動物 20
9. 動物實驗誘導 20
10. RNA 萃取套組 20
11. 反轉錄反應試劑 20
12. 即時定量聚合酶鏈鎖反應試劑 (Real-time PCR) 20
13. 免疫細胞分析 21
14. 酵素連結免疫吸附法 (ELISA) 21
15. 組織切片 21
16. 糞便 DNA 萃取套組 22
17. 短鏈脂肪酸及HPLC藥品 22
二、 儀器設備 22
三、 實驗方法 23
1. 實驗樣品製備 23
1-1. 來源 23
1-2. 胚芽乳酸桿菌樣品製備方法 23
1-2-1. 胚芽乳酸桿菌活菌樣品之製備 23
1-2-2. 熱裂解處理胚芽乳酸桿菌樣品 (Heat-killed probiotics, HK) 23
2. 樣品特性分析 23
2-1. 膠體滲透層吸法 (Gel Permeation Chromatography, GPC) 23
2-2. 硫酸根含量測定 24
2-3. 總醣含量測定(Total sugar content) 24
3. 細胞實驗 (In vitro) 24
3-1. 細胞培養 (Cell culture) 24
3-2. 細胞液之保存 25
3-3. 細胞液之活化 25
3-4. 細胞毒性測試 (Cytotoxicity test) 25
3-5. 肥大細胞脫顆粒試驗 25
3-6. 一氧化氮濃度分析 (NO assay) 26
4. 動物實驗 (In vivo) 26
4-1. 過敏性氣喘小鼠動物模式 26
4-2. 血液分析 27
4-3. 支氣管沖提液 (Bronchoalveolar lavage fluid, BALF) 分析 27
4-4. 分析組織中免疫細胞表現量 27
4-5. 酵素連結免疫吸附分析法 (Enzyme-linked immunoadsorbent assay, ELISA) 27
4-6. 分析組織中細胞激素之 mRNA 表現量 28
4-6-1. 抽取組織 total RNA 28
4-6-2. 反轉錄反應 (Reverse transcription, RT) 28
4-6-3. 即時聚合酶連鎖反應 (Real-time polymerase chain reaction) 28
4-7. 組織病理學切片與染色 29
4-7-1. 石蠟切片 29
4-7-2. 蘇木素-伊紅染色 (Hematoxylin and eosin stain, H&E stain) 29
4-8. 第三代基因定序（Full length 16S rRNA Generation Sequencing） 29
4-9. 短鏈脂肪酸分析 30
4-9-1. 糞便中短鏈脂肪酸萃取 30
4-9-2. 短鏈脂肪酸衍生化 30
4-9-3. 高液相層析法 (High performance liquid chromatography, HPLC) 流動相配置 31
4-9-4. 短鏈脂肪酸 HPLC 上機 31
4-10. 統計分析 31
第四章、 結果 32
一、 石蓴多醣組成分分析及體外細胞試驗 32
1. 石蓴水萃物之化學性質分析 32
2. 石蓴多醣與胚芽乳酸桿菌對於 P815 肥大細胞之影響 32
2-1. 石蓴多醣與熱裂解胚芽乳酸桿菌對 P815 細胞之細胞毒性 32
2-2. 石蓴多醣對 P815 細胞之細胞毒性 32
2-3. 熱裂解胚芽乳酸桿菌對 P815 細胞之細胞毒性 32
2-4. 石蓴多醣與熱裂解胚芽乳酸桿菌混合對 P815 細胞之細胞毒性 33
3. 石蓴多醣與熱裂解胚芽乳酸桿菌對 Compound 48/80 誘導 P815 細胞之脫顆粒效果 33
3-1. 石蓴多醣對Compound 48/80誘導 P815 細胞型態與脫顆粒情形 33
3-2. 熱裂解胚芽乳酸桿菌對Compound 48/80誘導 P815 細胞型態與脫顆粒情形 33
3-3. 石蓴多醣與熱裂解胚芽乳酸桿菌聯合給予對 Compound 48/80 誘導 P815 細胞型態與託顆粒情形 34
4. 石蓴多醣與胚芽乳酸桿菌對於 MH-S 肺泡巨噬細胞之影響 34
4-1. 石蓴多醣與熱裂解胚芽乳酸桿菌對 MH-S 細胞之細胞毒性 34
4-2. 石蓴多醣對 MH-S 細胞之細胞毒性 34
4-3. 熱裂解胚芽乳酸桿菌對 MH-S 細胞之細胞毒性 34
4-4. 石蓴多醣與熱裂解胚芽乳酸桿菌聯合給予對MH-S 細胞之細胞毒性 35
5. 石蓴多醣與熱裂解胚芽乳酸桿菌抑制 LPS 誘導MH-S 細胞發炎之效果 35
5-1. 石蓴多醣抑制 LPS 誘導MH-S 細胞發炎之效果 35
5-2. 熱裂解胚芽乳酸桿菌抑制 LPS 誘導MH-S 細胞發炎之效果 35
5-3. 石蓴多醣與熱裂解胚芽乳酸桿菌聯合給予抑制 LPS 誘導MH-S 細胞發炎之效果 35
二、 胚芽乳酸桿菌對過敏性氣喘小鼠之動物模式探討 36
1. 建立過敏性氣喘小鼠之動物模式 36
2. 每日體重紀錄及攝食變化 36
3. 組織器官分析 36
3-1. 肺臟組織重量分析 36
3-2. 脾臟組織重量分析 37
4. 發炎細胞計數 37
5. 石蓴多醣與胚芽乳酸桿菌對 OVA 誘導之小鼠脾臟中淋巴細胞的影響 37
6. 血清及支氣管肺泡沖提液中免疫球蛋白 E 及 Th1、Th2相關細胞因子分析 38
6-1. 血清免疫球蛋白 E 及 Th1、Th2 相關細胞因子分析 38
6-2. 支氣管肺泡沖提液免疫球蛋白 E 及 Th1、Th2 相關細胞因子分析 38
7. 肺組織細胞激素表達 39
7-1. Th2、Th1 相關細胞因子表達 39
7-2. Treg、Th17 相關細胞因子表達 40
8. 肺部組織切片 41
8-1. 蘇木素-伊紅染色 (Hematoxylin and eosin stain, H&E stain) 41
9. 糞便菌相 41
9-1. β 多樣性分析 41
9-2. ASVs 分析 41
10. 短鏈脂肪酸 42
第五章、 討論 43
1. 石蓴水萃物化學性質 43
2. 石蓴多醣與胚芽乳酸桿菌對於 P815 肥大細胞之影響 44
3. 石蓴多醣與胚芽乳酸桿菌對於 MH-S 小鼠肺泡巨噬細胞之影響 45
4. 石蓴多醣與胚芽乳酸桿菌於動物模式下改善皮質類類固醇之副作用 46
4-1. 石蓴多醣與胚芽乳酸桿菌於動物模式下其體重及攝食量之影響 46
4-2. 石蓴多醣與胚芽乳酸桿菌抗發炎之能力 46
4-3. 石蓴多醣與胚芽乳酸桿菌免疫調節之能力 47
5. 石蓴多醣與胚芽乳酸桿菌於動物模式下調節糞便菌相 49
第六章、 結論 51
第七章、 參考文獻 52
第八章、 圖 68
玖、表 91

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