臺灣博碩士論文加值系統

胃腺癌在台灣是國人十大癌症之一，而慢性萎縮性胃炎為胃腺癌的癌前病變徵兆。幽門螺旋桿菌 (Helicobacter pylori) 定殖於全球約50%人口之胃黏膜，並可能發展成萎縮性胃炎。抑制幽門螺旋桿菌所造成之發炎，成為相關研究之重要課題。
鳳仙花 (Impatiens balsamina L.) 為一種可食用之一年生草本植物，在亞洲也經常作為傳統中藥，本研究室過去於鳳仙花中純化出2-甲氧基-1,4-萘醌 (2-Methoxy-1,4-naphthoquinone, MeONQ)，並發現其具有良好之抗幽門螺旋桿菌活性，因此本研究欲探討在幽門螺旋桿菌感染之胃腺癌MKN45細胞中，MeONQ對於細胞發炎與細胞凋亡之調節機轉。
實驗結果顯示，MKN45細胞之細胞存活率，於MeONQ濃度低於3 μg/mL時均高於90%。幽門螺旋桿菌感染有效活化NF-κB核轉移與MAPK家族蛋白的磷酸化 (p<0.01, p<0.05)，顯著促進下游發炎蛋白COX-2表現與趨化素IL-8產生 (p<0.01)；於0.039 μg/mL之處理劑量，MeONQ可顯著降低p-JNK1/2、p-ERK1/2、COX-2蛋白表現與IL-8之產生 (p<0.05, p<0.01)，分別為感染組 (0 μg/mL) 之72%、25%、56%與73%。0.156 μg/mL之MeONQ可顯著抑制細胞核中NF-κB蛋白表現 (p<0.05)，為感染組 (0 μg/mL) 之53%。經幽門螺旋桿菌感染，有效活化MKN45細胞之Bax、Bak、Bcl-2、caspase-9與procaspase-3蛋白表現 (p<0.01, p<0.05) 並造成粒線體膜電位之有效降低 (p<0.01)； MeONQ之處理僅對Bcl-2蛋白表現造成顯著抑制 (p<0.01)，對於其他粒線體依賴途徑之凋亡相關因子，沒有造成顯著影響 (p<0.05)。以上結果顯示，MeONQ對發炎因子COX-2及趨化因子IL-8分泌造成之良好抑制效果，可能來自於對於上游轉錄因子NF-κB與AP-1活化的調控； COX-2的抑制調節抗凋亡蛋白Bcl-2的表現，然而並未因此促進細胞凋亡，此現象是否與其他信號傳遞路徑有關，需要經由進一步相關研究進行探討。

謝誌....................................................i
中文摘要................................................ii
Abstract..............................................iii
目錄....................................................v
附圖次.................................................ix
附表次..................................................x
圖次/表次...............................................xi
縮寫表................................................xii
壹、 前言............................................1
貳、 文獻整理.........................................2
一、 胃腺癌簡介.......................................2
二、 關於幽門螺旋桿菌.................................2
(一) 幽門螺旋桿菌簡介.................................3
(二) 幽門螺旋桿菌之致病性─毒力因子介紹..................3
(1) 空泡毒素 (vacuolating cytotoxin A, Vac A)........3
(2) 細胞毒素相關蛋白質 (cytotoxin associated gene A, Cag A)..................................................4
(3) 外膜發炎蛋白(outer membrane inflammatory protein, OipA)...................................................6
(4) 尿素酶 (urease).................................8
(5) 脂多醣 (lipopolysaccharide, LPS)................8
三、 幽門螺旋桿菌如何造成宿主細胞發炎反應─免疫反應途徑介紹.....................................................10
(一) PI3K途徑 (phosphatidylinositol-3-kinase pathway) .......................................................10
(二) Wnt途徑 (wingless type pathway)................11
(三) STAT3途徑(signal transducer and activator of transcription 3 pathway)...............................11
(四) NF-κB 途徑 (nuclear factor kappa-light-chain-enhancer of activated B cells pathway).................13
(五) MAPK途徑 (mitogen-activated protein kinase pathway)...............................................13
四、 感染幽門螺旋桿菌誘導之細胞發炎相關因子............15
(一) 細胞激素IL-6 (cytokine interleukin-6)...........17
(二) 誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS)........................................17
(三) 環氧合酶COX-2 (cyclooxygenase-2)................17
(四) 趨化素IL-8 (chemokine interleukin-8)...........18
(五) 血管內皮生長因子 (vascular endothelial growth factor, VEGF)..........................................18
五、 感染幽門螺旋桿菌誘導宿主細胞凋亡..................19
(一) 細胞凋亡與細胞壞死介紹...........................19
(二) 幽門螺旋桿菌使宿主細胞凋亡路徑....................19
六、 天然物對幽門螺旋桿菌誘發的發炎與凋亡反應之相關研究.....................................................24
七、 2-甲氧基-1,4-萘醌 (2-methoxy-1,4-naphthoquinone, MeONQ) 介紹............................................25
參、 研究目的........................................28
肆、 研究架構........................................29
伍、 材料與方法......................................30
一、 試驗化合物......................................30
二、 儀器...........................................30
三、 細胞株.........................................30
(一) 來源...........................................30
(二) 活化...........................................31
(三) 繼代...........................................32
(四) 保存...........................................32
四、 幽門螺旋桿菌菌株................................33
(一) 來源...........................................33
(二) 培養基配製......................................33
(三) 活化與培養......................................33
(四) 菌體增殖與培養..................................34
(五) 菌株保存........................................34
五、 以MTT測定法分析MeONQ對MKN45細胞存活率............34
六、 以JC-1染色分析MeONQ對感染幽門螺旋桿菌之MKN45細胞粒線體膜電位之影響..........................................35
(一) 藥品配製........................................35
(二) JC-1染色分析粒線體膜電位.........................35
七、 以西方墨點法分析MeONQ對感染幽門螺旋桿菌之MKN45細胞內蛋白表現之影響..........................................36
(一) 藥品配製........................................36
(二) 細胞貼附........................................40
(三) 幽門螺旋桿菌感染及MeONQ處理......................41
(四) 細胞分解液製備..................................41
(五) 蛋白質定量......................................42
(六) SDS-PAGE電泳分析................................42
(七) 免疫轉漬 (Immunoblotting) ......................43
(八) 顯影拍攝........................................44
八、 以ELISA分析MeONQ對感染幽門螺旋桿菌之MKN45細胞之IL-8產生量之影響............................................44
(一) 藥品配製........................................44
(二) 細胞貼附........................................46
(三) 幽門螺旋桿菌感染及MeONQ處理......................46
(四) 趨化素IL-8含量測定..............................46
九、 統計分析........................................47
陸、 結果與討論......................................48
一、 MeONQ對胃腺癌細胞MKN45之存活率...................48
二、 MeONQ對感染H. pylori ATCC 700824之MKN45細胞之信號傳遞路徑之影響............................................48
(一) MeONQ對感染H. pylori ATCC 700824之MKN45細胞MAPK家族之影響................................................48
(二) MeONQ對感染H. pylori ATCC 700824之MKN45細胞之NF-κB表現之影響..............................................50
(三) MeONQ對感染H. pylori ATCC 700824之MKN45細胞之發炎相關因子COX-2蛋白表現之影響................................50
(四) MeONQ對感染H. pylori ATCC 700824之MKN45細胞之趨化素IL-8之影響.............................................53
三、 MeONQ對感染H. pylori ATCC 700824之MKN45細胞之細胞凋亡相關因子表現之影響.....................................53
(一) MeONQ對感染H. pylori ATCC 700824之MKN45細胞Bcl-2家族蛋白表現之影響........................................56
(二) MeONQ對感染H. pylori ATCC 700824之MKN45細胞之粒線體膜電位 (ΔΨm) 之影響.....................................57
(三) MeONQ對感染H. pylori ATCC 700824之MKN45細胞之caspase-9及procaspase-3蛋白表現之影響...................57
四、 MeONQ對感染幽門螺旋桿菌之MKN45於細胞發炎與細胞凋亡之調節...................................................61
柒、 結論...........................................66
捌、 參考文獻 (References)...........................67

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