臺灣博碩士論文加值系統

Anandamide (N- arachidonoyl-ethanolamine)和PAF (platelet activating factor)是近來被鑑定出來的CB受體(cannabinoid receptor)內生性結合物，在我們的實驗中，分別去探討anandamide和2-O-methyl PAF在狗腎細胞(Madin Darby canine kidney cells)，對細胞訊息調節之作用機轉及在RAW 264.7巨噬細胞中，anandamide對發炎介質之調控作用。本研究是以Fura-2為鈣離子指示劑及WST-1，分別探討anandamide和2-O-methyl PAF等相關藥物對MDCK細胞內訊息調節之作用及對細胞生長影響，研究結果顯示anandamide(>5 ?嵱)和2-O-methyl PAF(> or =15 ?嵱)對狗腎細胞所引起之[Ca2+]i上升是來自於外鈣流入和鈣蓄池的內鈣釋出所致且anandamide造成細胞毒殺作用與濃度呈正相關性(100 ?嵱 – 200 ?嵱)。
另經由研究anandamide (100 ?嵱 – 400 ?嵱)在脂多醣體(Lipopolysaccharide； LPS)所誘發免疫細胞(RAW 264.7巨噬細胞)對發炎介質調控角色之探討中，anandamide對LPS所引發的nitrite、IL-6及IL-10等cytokines生成的影響和發炎過程中，anandamide是否具有清除ROS作用，另外對於影響LPS所引發PGE2生成和TNF受體角色亦一併探討。
總結而言，在我們的實驗中發現，anandamide和2-O-methyl PAF在狗腎細
胞，細胞訊息因子：cAMP、protein kinase A、phospholipase A2、Ca2+ signal都參與了調節之機轉；在LPS所誘發RAW 264.7巨噬細胞發炎反應中，anandamide具抗發炎之調控作用，可經由nitrite及IL-6等cytokines的抑制和ROS的清除以達到減緩發炎作用。
關鍵詞：anandamide，2-O-methyl PAF，狗腎細胞，脂多醣體，RAW264.7吞噬細胞，細胞激素

Recently, the involvement of anandamide and PAF in endogenous cannabinoid system have been suggested. The present study was to investigate the inflammatory regulation of anandamide and its related molecular mechanisms. Efforts were made to examine whether anandamide and 2-O-methyl PAF altered Ca2+ levels and caused Ca2+-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. In MDCK cells, anandamide (>5 ?嵱) and 2-O-methyl PAF (> or =15 ?嵱) induced [Ca2+]i rises by causing Ca2+ release from endoplasmic reticulum and Ca2+ influx from extracellular space. Furthermore, anandamide (100 ?嵱 – 200 ?嵱) can cause Ca2+-dependent cytotoxicity in a concentration-dependent manner.
In the endotoxic model, the effects of anandamide on nitrite, tumor necrosis factor-alpha (TNF-α) and interleukins (IL)-6, and -10 formation in lipopolysaccharide (LPS) -treated RAW 264.7 macrophages and the possible anti-inflammatory mechanisms involved were also clarified. RAW 264.7 macrophages were simultaneously incubated with anandamide at final concentration of 100- 400 (?嵱) and LPS (1 microg/ml) for 24 h on a mechanical shaker.
These results suggest that anandamide and 2-O-methyl PAF may exert cell signaling regulation, the cAMP、protein kinase A、phospholipase A2 and Ca2+ signal were involved the regulation in MDCK cells. Anandamide and 2-O-methyl PAF induced effects apprar to be regulated by different second messengers. In LPS stimulated RAW 264.7 macrophages, anandamide regulates inflammatory responses by a mechanism that involves the attenuation of nitrite induction, free radical generation and modulation of inflammatory cytokines.
Keywords anandamide, 2-O-methyl PAF , MDCK cells , LPS , RAW macrophages, Cytokines

摘要…………………………………………………………….1
縮寫表………………………………………………………….7
壹.緒論………………………………………………………..10
貳.文獻探討………………………………………………......13
(一) 大麻素與大麻素受體……………………………..… 13
(二) 細胞訊息調控……………………………………..… 17
(三) 發炎因子與作用機制………………………………...26
(四) 細胞內活性氧物質(ROS)與細胞凋亡………………33
(五) NO對細胞凋亡的調控………………………….....…34
參.材料與方法……………………………………………..… 48
(一) 細胞存活率分析
1. 細胞培養………………………………...…………50
2. 細胞檢量線圖分析……………………...…………51
3. 細胞存活率測定方法……………….……………..51
(二) 細胞訊息因子之測定……….……………...………..52
(三) 發炎因子之測定……………………….…………….53
(四) 細胞內活性氧物質之測定……...….………………..53
(五) 大麻素對細胞程式死亡相關訊息之分子調節機轉
1.細胞之TNF-α含量百分比測定………………..54
2.NO細胞含量之測定(Griess Regent)……………54
(六) 統計分析……………………………………………..55
肆.結果………………………………………………………..56
伍.討論………………………………………………………..62
陸.參考文獻…………………………………………………..66
柒.實驗結果圖表……………………………………………..79
捌.會議及論文發表…………………………………………..99

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