僵直性脊椎炎是一種自體免疫疾病，主要造成薦腸關節、脊椎關節發炎、心血管、肺部和腸胃道侵犯的特徵。HLA-B27在僵直性脊椎炎病因上扮演一個重要的角色，但是調控機轉仍然不是很清楚。傳統中草藥治療僵直性脊椎炎已經超過三千年，我們篩選超過60種以上中草藥及生物活性成分來作為治療僵直性脊椎炎可能藥物。許多研究指出克雷伯氏肺炎桿菌和僵直性脊椎炎有相當的關係，但是致病機制仍然不是很清楚，因此我們使用去莢膜克雷伯氏肺炎菌來誘發表現HLA-B27的CA46 B淋巴球母細胞、不含HLA-B27的U937單核球細胞可以產生內質網壓力，我們先前的實驗指出，隨著劑量增加，去莢膜克雷伯菌能夠過度調控內質網壓力指標蛋白質BiP和Calreticulin表現增加。我們篩選超過60種中藥及生物活性成分中，並且發現小蘗鹼 (berberine)、牛磺酸 (taurine)、雞血藤萃取物和穿心蓮萃取物能夠保護去莢膜克雷伯氏肺炎桿菌誘發U937單核球細胞、CA46 B淋巴球細胞和健康者身上週邊血液單核球細胞的內質網壓力。去莢膜克雷伯氏菌會誘發健康者週邊血液單核球細胞產生IL-1β、IL-6和TNF-α表現量增加，小蘗鹼則明顯降低TNF-α表現而牛磺酸和穿心蓮也能稍微降低TNF-α表現。小蘗鹼保護去莢膜克雷伯氏肺炎桿菌誘發U937單核球細胞與週邊血液單核球細胞產生內質網壓力主要透過抑制p38與JNK的活化。去莢膜克雷伯氏肺炎桿菌能誘發內質網壓力和發炎而小蘗鹼可能可以治療因為內質網壓力所造成的疾病。

Abstract
Ankylosing spondylitis is an autoimmune disease characterized by sacroiliac joints、spinal join arthritis、cardiovascular system、pulmonary system and gastrointestinal system involvement. HLA-B27 plays an important role in the etiology of ankylosing spondylitis but the modulation remain unclear. Many traditional herbs were used in the treatment of ankylonsing spondylitis for more than three thousand years. More than 60 kinds of traditional herbs and bioactivity agent are selected for the study of ankylosing spondylitis. Many studies have reported that Klebsiella pneumoniae (KP) is associated with ankylosing spondylitis but the mechanism of pathogenesis is remained unclear. The endoplasmic reticulum stress would be induced upon exposure of decapsulated KP in HLA-B27 expression B lymphoblast CA46 cells and non-HLA-B27 expression U937 monocytes. Our previous studies demonstrated that decapsulated KP could up-regulate BiP/Grp78 and calreticulin expressions in a dose- and time-dependent manner. We screen more than 60 kinds of traditional herbs and bioactivity agents and we found that berberine，taurine，extracts of Andrographis paniculata Nees. and Spatholobus suberectus Dunn. provided protection activities against decapsulted KP-induced ER stress in U937 monocytes、B lymphoblast CA46 cells and PBMC of normal individuals. We also found a significantly increase of IL-1β、IL-6 and TNF-α proinflammatory cytokine by PBMC obtained from normal individual under decapsulated KP treatment. Interestingly，significant suppression of TNF-α secretion under the treatment of berberine was found，and lower levels of suppressive effects under the treatment of taurine or extract of Andrographis paniculata Nees. are also found. In addition，berberine could suppress activations of p38 and JNK stimultaneously in U937 cells and PBMC upon decapsulated KP treatment. Non-capsule components of KP might initiate ER stress & inflammation，and berberine might has the potential for treating disease induced via ER stress.

目錄
中文摘要……………………………………………….……………………………....i
Abstract…………………………………………………..………………………..….iii
致謝…..………………………………………………….…………………….………v
目錄……………………………………………………………………………...…..vii
縮寫表……………………………………………………..……………………….…ix

第一章 緒論…………………………………………………………………………1
一、 僵直性脊椎炎…………………………………………………………2
二、 內質網壓力……………………………………………………………6
三、 克雷伯氏肺炎桿菌…………………………………..………………13
四、 傳統中醫對僵質性脊椎炎之看法………………………………..…15
五、 研究目的…………………………………………..…………………22
六、 研究執行目標. …………………………………..…………………..22

第二章 材料與方法………………………………………………………………..24
一、 K. pneumoniae 培養與去莢膜 (K. pneumoniae culture and decapsulation)………………………………………….……………..25
二、 分離中藥有效成分…………………………………….…………….27
三、 細胞培養……………………………………………………………..29
四、 蛋白質電泳與西方墨點法…………………………………………..32
五、 分離週邊血液單核球細胞 (peripherial blood mononuclear cells, PMBC isolation)…..…………………………………………….……38
六、 免疫酵素沉澱法 (Enzyme-linked Immunosorbant Assay, ELISA)...40

第三章 結果………………………………………………………………….…….43
一、 中藥對細胞的毒性測試…………………………………….……….44
二、 去莢膜克雷伯氏肺炎桿菌引發各類免疫細胞產生內質網壓力…. 45
三、 中藥能夠抑制去莢膜克雷伯氏肺炎桿菌所引發的各類免疫細胞產生內質網壓力………………………………………………………..46
四、 小蘗鹼、牛磺酸和穿心蓮能抑制去莢膜克雷伯氏肺炎桿菌所引發的各類免疫細胞產生內質網壓力……………………………………..46
五、 去莢膜克雷伯氏肺炎桿菌引發周邊血液單核球細胞產生內質網壓力..........................................................................................................47
六、 中藥能夠壓抑去莢膜克雷伯氏肺炎桿菌所引發的週邊血液單核球細胞內質網壓力..……………………………………………………48
七、 去莢膜克雷伯氏肺炎桿菌引發周邊血液單核球細胞產生促發炎細胞激素的上升 …………..…………………………………………..49
八、 中藥能夠壓抑或促進去莢膜克雷伯氏肺炎桿菌所引發的產生週邊血液單核球細胞促發炎性細胞激素的改變 ………………………50
九、 小蘗鹼能夠抑制U937細胞受到去莢膜克雷伯氏肺炎桿菌刺激後活化MAPK路徑……………………………………………………..…51
十、 週邊血液單核球細胞受到去莢膜克雷伯氏肺炎桿菌刺激後活化MAPK路徑，而小蘗鹼能夠抑制此條路徑……………………..…52

第四章 討論………………………………………………….…………………….53
一、 去莢膜克雷伯氏肺炎桿菌的哪些成分可能是造成免疫細胞產生內質網壓力的致病因子……………………….………………………54
二、 去莢膜克雷伯氏肺炎桿菌刺激U937產生內質網壓力的下游路徑及小蘗鹼的機轉探討……….….……..…….………………………….55

第五章 結論.……………………………………………………………………….56

參考文獻………………………………………………….……………………....57-64
圖………………………………………………………….……………………....65-77
表………………………………………………………….……………………....78-80
附錄……………………………………………………….………………………81-85

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