流感病毒 (Influenza virus, IFV) 是一種高度傳染性疾病，對全球健康產生重大影響。這種病毒是一種主要的呼吸道病原體，會導致高發病率和死亡率，尤其是在免疫功能低下的宿主中。目前國内使用的抗流感病毒藥劑主要為克流感及瑞樂沙，然而不良的副作用限制了這些藥物在治療流感中的用途，因此需要去開發新的抗流感藥物。益生菌等天然成分已經過多次研究，並已被證明是一種安全的替代預防和治療方法，可通過刺激誘導免疫途徑來調節免疫反應。發現益生菌抗病毒的文獻主要分為兩種方式-鼻腔給予方式及口服給予方式，但文獻少對植物乳桿菌 (Lactobacillus plantarum) 做兩種給予方式的比較及抗病毒之機制對比，且有文獻指出免疫調節特性取決於菌株。因此，本研究的目的是探討通過口服或鼻腔給予益生菌的方式對抗A型流感病毒感染的保護作用和機制。在細胞病變抑制實驗及病毒斑抑制試驗中，將植物乳桿菌在預防及治療的條件下添加於細胞，皆無法提升犬腎細胞的存活率。在動物實驗中，分別以管餵給予和鼻腔給予1 х 109 cell/mice 的活菌Live L. plantaru (LP)及10 mg/kg/day 的熱裂解菌heat-killed L. plantarum (HK) 和上清液supernatant (SN)進行預防試驗，並使用兩倍LD50半致死劑量之病毒液噴霧感染BALB/c小鼠。實驗結果顯示口服和鼻內給予植物乳桿菌可調節免疫系統，其中包括活化NK細胞及分化Th1輔助型T細胞來提升先天及適應性免疫，並抑制促發炎細胞因子IL-6、TNF-α 及 MCP-1的分泌以減少小鼠肺部的發炎損傷效果，從而提高小鼠的存活率。而鼻腔給予活菌更可以促進毒殺型T細胞及IgA免疫球蛋白抗體減少病毒的擴散，口服給予熱裂解菌則刺激NK活化及IgG減少病毒擴散至全身。結合上述結果，本篇植物乳桿菌以不同給予方式各有不同機制來調節免疫反應，並顯示出對A型流感病毒感染的保護和預防作用。

Influenza virus (IFV) is responsible of a highly contagious disease that has a substantial impact on global health. This virus is a major respiratory pathogen that causes a high degree of morbidity and mortality, especially in immunocompromised hosts. At present, the anti-influenza virus drugs used in domestic are mainly Tamiflu and Relenza. However, the adverse side effects limit the use of these drugs in the treatment of influenza. Therefore, it is necessary to develop new anti-flu drugs. Natural components like probiotics have been severally studied and have been proved to be a safe alternative prophylactic and therapeutic approach for modulating immune responses via the induction of the newly described process of trained immunity. The anti-influenza virus study in probiotics is mainly intranasal administration and oral administration, but it rarely compared the administration methods and the antiviral mechanisms of Lactobacillus plantarum. Research have suggested that the immunoregulatory functions is based on the strain. Therefore, the purpose of this study was that using oral or intranasal administration to study of the protective effects and mechanism of probiotics against influenza A virus infection. In the cytopathic inhibition test and the plaque inhibition assay, heat-killed L. plantarum (HK) and supernatant (SN) couldn't increase the survival rate of MDCK cells under the conditions of pre-treatment and post-treatment. In animal experiments, pre-treated with oral administration and intranasal administration of Live L. plantarum (LP) at 1 х 109 cell/mice, HK and SN at 10 mg/kg/day. BALB/c mice were infected with H1N1 aerosol at a dose of 2 times of the LD50. In vivo study, it was found that oral and Intranasal administration of Lactobacillus treatment regulated the immune system, included activation of NK cells and T cells for strengthen innate and adaptive immunity, diminished the pro-inflammatory cytokines like as IL-6, TNF-α, MCP-1 in the lung tissue to enhancing the mice survival rate. The intranasal administration of Live L. plantarum can promote cytotoxic T cells and IgA antibodies to reduce the adhesion of the virus, while the oral administration of heat-killed L. plantarum stimulates NK activation and IgG antibodies to reduce the spread of the virus to the whole body. Combining the above results, Lactobacillus plantarum in different ways of administration has different mechanisms to modulate the immune response and exhibit both protective and prophylactic effects against influenza A virus infection.

摘要 Ⅱ
Abstract Ⅲ
目錄 Ⅳ
圖目錄 Ⅷ
第一章、前言 1
第二章、文獻回顧 2
壹、 流感病毒 2
一、 流感病毒之流行病學 2
二、 流感病毒之流行史 2
三、 流感病毒之簡介 3
四、 流感病毒的複製過程 4
五、 流感病毒的致病機制 5
六、 流感病毒感染後之免疫反應 5
七、 流感病毒之臨床病徵 6
八、 流感病毒的治療與預防 6
貳、 益生菌 (Probiotics) 7
一、 益生菌的介紹 7
二、 益生菌的作用機制 7
三、 益生菌之功效 8
第三章、實驗設計 11
壹、 細胞實驗 (In vitro) 11
貳、 動物實驗 (In vivo) 12
第四章、材料與方法 14
壹、 實驗材料 14
一、 實驗菌株 14
二、 細胞株 14
三、 病毒株 14
四、 細胞培養 14
五、 細胞存活率試劑 14
六、 病毒斑試驗 15
七、 一氧化氮試驗 15
八、 實驗動物 15
九、 RNA萃取套組 15
十、 反轉錄反應試劑 15
十一、 即時定量聚合酶鏈鎖反應試劑 15
十二、 酵素免疫分析套組 16
十三、 組織切片 16
貳、 儀器設備 16
參、 實驗方法 17
一、 實驗樣品製備 17
1. 來源 17
2. 植物乳桿菌樣品製備方法 17
二、 流感病毒增殖培養 17
三、 細胞實驗 (In vitro) 18
1. 細胞活化 (Cell thawing) 18
2. 細胞培養 (Cell culture) 18
3. 細胞保存 (Cell freeze) 19
4. 細胞毒性試驗 (Cytotoxicity) 19
5. 病毒斑試驗 (Plaque assay) 19
6. 細胞病變抑制試驗 (Cytopathic effect inhibition assay) 19
7. 病毒斑抑制試驗 (Plaque inhibition assay) 20
8. 一氧化氮濃度分析 (NO assay) 20
四、 動物實驗 (In vivo) 21
1. 流感病毒感染小鼠之動物模式 21
2. 血液分析 (Hematology analysis) 22
3. 肺支氣管沖提液 (Bronchoalveolar lavage fluids, BALF) 22
4. 免疫細胞分析 22
5. 偵測肺組織中 RNA 表現量 22
6. 酵素結合免疫吸附分析法 (Enzyme-linked immunosorbent assay, ELISA) 24
7. 肺組織病理切片分析 (Histopathology analysis) 24
五、 統計分析 (Statistics analysis) 24
第五章、結果 25
壹、 植物乳桿菌抗 A 型流感病毒 (H1N1) 感染與抗發炎之細胞模式評估 25
一、 熱裂解植物乳桿菌及上清液對於 MDCK 細胞之細胞毒性 25
二、 熱裂解植物乳桿菌及上清液對於 H1N1 感染 MDCK 細胞的存活率影響 25
三、 熱裂解植物乳桿菌及上清液抑制 H1N1 之病毒斑生成 25
四、 熱裂解植物乳桿菌及上清液對於 RAW264.7 細胞之細胞毒性 26
五、 熱裂解植物乳桿菌及上清液抑制 LPS 誘導RAW264.7 細胞發炎之效果 26
六、 熱裂解植物乳桿菌及上清液對於 MH-S 細胞之細胞毒性 26
七、 熱裂解植物乳桿菌及上清液抑制 LPS 誘導MH-S 細胞發炎之效果 27
貳、 利用動物模式探討植物乳桿菌樣品對於 A 型流感病毒 (H1N1) 感染之影響 27
一、 確定 H1N1 噴霧感染小鼠之劑量 27
二、 植物乳桿菌樣品於感染 H1N1 小鼠之存活率及體重影響 27
三、 植物乳桿菌樣品於感染 H1N1 小鼠之健康指數影響 28
四、 植物乳桿菌樣品對感染H1N1小鼠之肺發炎損傷情形 29
五、 植物乳桿菌樣品對於感染H1N1之血液分析 29
六、 植物乳桿菌樣品對於感染H1N1小鼠肺組織中細胞因子及趨化因子基因表現影響 30
七、 植物乳桿菌樣品對於感染 H1N1 小鼠血液及肺沖提液中細胞激素抗原表現影響 31
八、 植物乳桿菌樣品對於感染 H1N1 小鼠脾臟中淋巴細胞的影響 32
九、 植物乳桿菌樣品對於感染 H1N1 小鼠肺組織之組織病理學表現情形影響 32
第六章、討論 33
壹、 植物乳桿菌樣品萃取的三種型態 33
貳、 以體外試驗探討植物乳桿菌樣品抗 A 型流感病毒 (H1N1) 感染與抗發炎之評估 34
參、 以IFV感染BALB/c 小鼠之動物模型 35
肆、 利用動物模式探討探討植物乳桿菌樣品之保護效果 36
一、 植物乳桿菌樣品對於感染 H1N1 小鼠之存活率影響 36
二、 植物乳桿菌樣品對於感染 H1N1 小鼠免疫系統保護之影響 37
第七章、結論 41
參考文獻 42
第八章、圖表 58

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