臺灣博碩士論文加值系統

COX-2（cyclooxygenase-2）為一種受到外來刺激時的誘導型酵素，而COX-2的反應會產生前列腺素E2 (prostaglandin E2， PGE2)，引發發炎反應。而cytokine參與免疫反應和發炎反應過程，其中IL-1β(interlukine-1β)是一種十分重要的免疫調節因子，在多種細胞中，經發炎、感染、內毒素所產生。而巨噬細胞經刺激後也會產生大量的自由基，來進行多種發炎、免疫過程的重要介質。
細胞中內質網鈣離子蓄池（ER Ca2+ store）在各種生理作用中皆扮演了重要的角色。本研究中將以一種內質網鈣離子蓄池不可逆性的抑制劑 TG（thapsigargin），處理老鼠巨噬細胞RAW264.7，以相同培養液，不同模式處理細胞，並以LPS（Lipopolysaccharide）加以刺激細胞，利用western blotting的方法來看COX-2的表現量，並且以酵素免疫分析法測量PGE2的濃度和IL-1β產量，最後用呈色反應的方式測量NO的產量，螢光試劑測量H2O2。藉以探討以TG處理細胞，抑制內質網Ca2+-ATPase後，或以其他增加胞內鈣離子之物質，當中COX-2在細胞中的表現量及COX-2的表現，對於細胞激素分泌的影響與自由基產量變化相關性。.

Cyclooxygenase-2 (COX-2), an inducible form enzyme, is expressed after external stimuli, then produces prostaglandin E2 (PGE2) to propagate inflammation processes. The pro-inflammatory cytokines are secreted by a variety of cells in response to infection, inflammation and endotoxin- activated, which interleukin-1β (IL-1β) is major immune regulation factor. In this study, murine macrophage RAW 264.7 cells were pretreated with thapsigargin (TG), an ER-Ca2+ ATPase irreversible inhibitor, for 1 hr then wash out and changed to fresh medium, and immediately stimulate with 5 μg/ml LPS. The observation of the expression of COX-2 and protein kinase C (PKC) isoforms were depended by Western blotting. Usage of Enzyme immunoassay detected PGE2 and IL-1β production, Usage of Griess reagent measured nitrite oxide (NO) production. Usage of electrophoresis mobility shift assay (EMS) detected the activation of AMP response element binding protein (CREB) transcription factor. H2O2 production was detected by flowcytometer by DCF-DA.

The results showed that cells were pretreated with TG for 1 hrs and then wash out applied with LPS. The expression of COX-2 was significantly increased. The production of PGE2 and IL-1β also were increased. However, the time duration of production of IL-1β was later after PGE2. The pretreatment with TG was also increased the activation of PKC-ζ and CREB transcription factor. On the other hand, usage of Ca2+ ionophores: A23187, ionomycin, or DBHQ, a reversible ER Ca2+- ATPase inhibitor, could not mimic TG to increase COX-2 expression and PGE2 production.

Base on above analytic data indicated that when RAW 264.7 cells were blocked ER Ca2+- ATPase by TG then treated with LPS could enhance the expression of COX-2 and the production of PGE2. The increase of intracellular calcium or the production of IL-1β may not be the major role to induce these effects. The inhibition of ER Ca2+ ATPase may be the major role that enhanced LPS-activate PKC-ζand CREB to increase the expression of COX-2 and the production of PGE2.

目 錄
頁次
目錄
圖目錄
表目錄
縮 寫 表
第一章 緒論
第二章 研究材料與方法
2.1 細胞培養
2.2 細胞毒性測試
2.3 自由基之測定
2.4 IL-1β之測定
2.5 PGE2之測定
2.6 蛋白質之萃取及分析
2.7 轉錄因子 (CREB)的測定
2.8 實驗藥品
第三章 研究結果
3.1 Thapsigargin 對巨噬細胞誘發一氧化氮（NO）的影響
3.2 TG對巨噬細胞 LPS 所誘發COX-2蛋白表現之影響
3.3 TG、DBHQ、A23187 及 ionomycin 對巨噬細胞 LPS 所誘發 COX-2 蛋白表現之影響
3.4 TG、DBHQ、A23187 及 ionomycin 對巨噬細胞 LPS 所誘發 PGE2 之影響
3.5 處理 Meloxicam 對巨噬細胞誘發 PGE2 之影響
3.6 TG、DBHQ、A23187 及 ionomycin 對巨噬細胞誘發IL-1β之影響
3.7 TG、DBHQ、A23187 及ionomycin 對於 CREB 活化之影響
3.8 TG 對 PKCζ 活化之影響
3.9 TG 對巨噬細胞誘發H2O2 之影響
3.10 以不同的方式對細胞處理 TG ，對 NO、COX-2、PGE2 及 IL-1β 的影響
第四章 討 論
參考文獻









圖目錄
圖一 Thapsigargin （TG）前處理後減少LPS 刺激巨噬細胞誘發NO 之產量 32
圖二 TG 影響 LPS 誘發巨噬細胞 NO 產量及細胞存活率 33
圖三 DBHQ, A23187 及ionomycin 影響LPS 誘發巨噬細胞 NO 產量及細胞存活率 34
圖四 TG 對於 LPS 誘發巨噬細胞 COX-2 蛋白表現之影響 35
圖五 不同TG 濃度對於LPS誘發巨噬細胞COX-2 蛋白表現之影響 36
圖六 DBHQ, A23187 及 ionomycin 對LPS誘發巨噬細胞COX-2之影響 37
圖七 TG 對巨噬細胞LPS所誘發PGE2 之影響 38
圖八 TG, DBHQ, A23187 及 ionomycin 對巨噬細胞LPS所誘發PGE2 之影響 39
圖九 以 COX-2 抑制劑 meloxicam（MI）後對PGE2之影響 40
圖十 以 COX-2 抑制劑 meloxicam（MI）後對NO之影響 41
圖十一 TG, DBHQ, A23187 及 ionomycin 處理後影響LPS 刺激巨噬細胞內CREB的活化 42
圖十二 TG 處理後影響LPS 刺激巨噬細胞內PKCζ的活化 43
圖十三 以不同的方式處理TG對於LPS誘發細胞NO及細胞存活率的影響 44
圖十四 以不同的方式處理DBHQ, A23187, ionomycin對於LPS誘發細胞NO及細胞存活率的影響 45
圖十五 以不同的方式處理TG對於LPS誘發COX-2蛋白表現的影響 46
圖十六 以不同方式處理TG後對於LPS誘發巨噬細胞PGE2之影響 47
圖十七 COX-2 生成 PGE2 路徑圖 48


























表目錄

表一 TG 對巨噬細胞誘發 IL-1β分泌之影響 49
表二 TG, DBHQ, A23187 及 ionomycin對巨噬細胞誘發 IL-1β分泌之影響 50
表三 以COX-2 抑制劑 meloxicam（MI）後對於LPS誘發IL-1β之影響 51
表四 TG, DBHQ, A23187 及 ionomycin對於巨噬細胞於DCF螢光強度之影響 52
表五 以不同方式處理TG後對於LPS誘發巨噬細胞IL-1β之影響 53

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