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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃德富 | |
dc.contributor.author | Ting-Yun Huang | en |
dc.contributor.author | 黃亭雲 | zh_TW |
dc.date.accessioned | 2021-06-15T01:14:08Z | - |
dc.date.available | 2011-09-15 | |
dc.date.copyright | 2009-09-15 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-28 | |
dc.identifier.citation | REFERENCES
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42459 | - |
dc.description.abstract | 發炎反應即個體遭遇到外界有害刺激,像是病原菌、有害化學物質、傷口感染、組織受損時,體內所產生的自然防禦機制。適當的發炎反應得以協助人體抵擋來自外界的傷害,清除病原菌,藉以恢復健康。然而過度甚而失去控制的強烈發炎反應,其產生的發炎相關激素與機制,往往會在體內不斷累積並放大反應,最終於器官組織造成損害,引發一系列的相關疾病。近來許多研究發現,發炎反應的確與關節炎、多發性硬化、氣喘、動脈硬化等慢性疾病有所相關,甚至某些癌症的發生也歸咎於持續而激烈的發炎反應。正因如此,研究開發有效的抗發炎藥物,減輕患者的發炎症狀,並試圖終止強烈的發炎反應,也成了現今許多專家學者與藥廠努力的方向與動力。
在實驗室初步篩藥過程中,發現一化學合成物質pcpc07f2具有抑制巨噬細胞釋放細胞激素的能力,引發我們進一步探究其抗發炎能力與機轉的興趣。首先確認了實驗中所用濃度並不會影響噬菌細胞株的生存率。接著在抑制噬菌細胞受到LPS 活化後釋放發炎性細胞激素之測試中,我們發現pcpc07f2在10μg/ml (約30μM)即能有效抑制由LPS 100ng/ml在原生性小鼠巨噬細胞、細胞株RAW264.7巨噬細胞、亦或細胞株THP-1單核細胞所引發之TNF-α與IL-6釋放,且此抑制作用呈現明顯的濃度相關性。除此之外,對於LPS 100ng/ml活化細胞株RAW264.7巨噬細胞後,其產生之移動現象(migration) 以及大量釋放nitric oxide的發炎反應,pcpc07f2也都能在10μg/ml (30μM)時達抑制作用,同樣此抑制作用呈現明顯的劑量相關性。最後,延伸pcpc07f2之抗發炎能力,將其運用至LPS誘發之敗血症動物模式中,結果發現pcpc07f2 (8μg/g)能有效降低敗血症小鼠血清中的發炎細胞激素含量(TNF-α與IL-6),並明顯降低LPS誘發敗血症小鼠之死亡率。 而對於LPS刺激活化巨噬細胞後所產生的下游訊息傳遞鏈之中,我們檢視了MAPK 路徑中的三種重要激酶:p-38、ERK、JNK的磷酸化現象,以及NF-κB路徑中重要調控因子: IκB的降解情形 (degradation)。研究結果發現,在給予pcpc07f2之後,p-38、ERK、JNK這些MAPK重要激酶的磷酸化都有被抑制的現象,而抑制NF-κB活化的IκB,其降解情形也可被pcpc08f2所回復(reverse),進而抑制NF-κB的活化。此外,我們也使用了MAPK和NF-κB inhibitor來研究兩種訊息傳遞路徑和發炎激素之間的關聯性,發現它們可以抑制LPS刺激活化之巨噬細胞釋放發炎性細胞激素與nitric oxide。 綜合以上所述,我們可以推測pcpc07f2可能經由抑制MAPK路徑中的三種重要激酶p-38、ERK、JNK的磷酸化,以及抑制NF-κB此轉錄因子的活化,來進行其抑制發炎的能力。當然,詳細的作用機轉還有待更多方面的實驗進一步來探討。 | zh_TW |
dc.description.abstract | Macrophages play a vital role in inflammation and innate immune responses. Foreign pathogens recognized by macrophages trigger a serial of signaling cascades upon ligand-receptor stimulation, then resulting in releasing several pro-inflammatory and anti-inflammatory cytokines, and activating other immune cells. In this study, we used lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall for activating macrophages, as an experimental model. The pro-inflammatory cytokines, such as TNF-α and IL-6 increased significantly after LPS stimulation as measured by ELISA. We found pcpc07f2, a synthetic benzimidazole compound, concentration-dependently (10~90μM) inhibited LPS (100ng/ml)-induced TNF-α and IL-6 release in macrophages (RAW 264.7 cells), and the inhibition appeared more obvious in primary macrophages. However, pcpc07f2 did not affect the cell viability. Regarding the anti-inflammation effects of pcpc07f2 on LPS-induced septic animal model, we also found that pcpc07f2 significantly reduced pro-inflammatory cytokines release and also reduced LPS-induced mortality in a dose-dependent manner. Furthermore, pcpc07f2 also inhibited macrophage migration and NO production induced by LPS. We further examined if pcpc07f2 affects the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in LPS-induced RAW 264.7 cells by Western blotting analysis. Pcpc07f2 concentration-dependently inhibited LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2), JNK, and p38 phosphorylation, and suppressed LPS-induced NF-κB activation, suggesting that pcpc07f2 decreased TNF-α, IL-6 and NO production via the inactivation of ERK1/2, JNK, and p38 ,and NF-κB signal pathway. Taken together, we suggest that pcpc07f2 may be used as therapeutic approach for the treatment of inflammatory diseases. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:14:08Z (GMT). No. of bitstreams: 1 ntu-98-R96443001-1.pdf: 1850180 bytes, checksum: f93007a43fc60b6a9c06f215268f953c (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 目 錄
中文摘要…………………………………………………………………………. i Abstract ..………………………………………………………………………..... iii Abbrevation table .................................................................................................... v Chapter 1 Introduction ................................................................................................1 1.1 Inflammation ...........................................................................................1 1.2 Monocyte/macrophage ............................................................................2 1.3 Cytokine ...................................................................................................3 1.3.1 Tumor Necrosis Factor-α (TNF-α) .................................................5 1.3.2 Interleukin-6 (IL-6) .........................................................................6 1.4 NO ............................................................................................................7 1.5 The signaling pathways of lipopolysaccharide (LPS) ..............................8 1.5.1 Activation of NF-κB and regulation of gene expression .................9 1.5.2 Phosphorylation of mitogen-actived protein kinases (MAPKs) signaling pathway ............................................................................11 1.6 Sepsis .......................................................................................................14 1.7 Cancer and inflammation ........................................................................15 1.8 Aims of this thesis....................................................................................15 Chapter 2 Materials and methods ................................................................................26 2.1 Materials ....................................................................................................26 2.2 Cell cultures ...............................................................................................27 2.2.1 Cell culture – RAW 264.7 macrophage cell ...................................27 2.2.2 Cell culture – Murine peritoneal macrophage cell .........................27 2.2.3 Cell culture – Human THP-1 monocytic leukemia cell .................28 2.3 Cell viability assay ....................................................................................28 2.4 Nitric oxide assay ......................................................................................29 2.5 Cytokine assays .........................................................................................30 2.6 Migration assay .........................................................................................31 2.7 Western blot analysis .................................................................................32 2.8 Thromboxane A2 synthase, COX and other kinases or enzymatic activity assay ..........................................................................................................33 2.9 Animal model ............................................................................................33 2.9.1 Animals ...........................................................................................33 2.9.2 LPS challenge .................................................................................33 2.9.3 Mice whole blood and plasma collection .......................................33 2.9.4 Measurement of cytokine levels .....................................................34 2.10 Statistical analysis ...................................................................................34 Chapter 3 Results ........................................................................................................35 3.1 The effects of pcpc07f2 on LPS-stimulated pro-inflammatory TNF-α production in phagocytes ..........................................................................35 3.2 The effects of pcpc07f2 on LPS-stimulated pro-inflammatory cytokine IL-6 production in phagocytes ..................................................................35 3.3 The effects of pcpc07f2 on cell viability in murine macrophage cells in vitro and ex vivo ........................................................................................36 3.4 The inhibition of Pcpc07f2 on NO production in LPS-induced RAW 264.7 macrophage cells .......................................................................................36 3.5 Pcpc07f2 inhibits the migration of LPS-activated RAW264.7 macrophage cells ............................................................................................................37 3.6 The effects of pcpc07f2 on the LPS-stimulated activation of mitogen-activated protein (MAP) kinases ...............................................38 3.7 Pcpc07f2 inhibits IκB degradation in LPS-induced RAW264.7 macrophages ............................................................................................39 3.8 The effects of MAPKs-specific inhibitors and NF-κB inhibitor (PDTC) on the production of cytokines TNF-α and IL-6 in LPS-stimulated RAW264.7 macrophages ........................................................................39 3.9 The effects of MAPKs-specific inhibitors and NF-κB inhibitor (PDTC) on the production of NO in LPS-stimulated RAW264.7 macrophages .......40 3.10 Pcpc07f2 inhibits LPS-induced pro-inflammatory cytokines production such as TNF-α and IL-6 in vivo ...............................................................41 Chapter 4 Discussion ...................................................................................................62 Chapter 5 Conclusion ..................................................................................................67 Chapter 6 In the future .................................................................................................69 References ...............................................................................................................70 | |
dc.language.iso | en | |
dc.title | Pcpc07f2抑制LPS活化噬菌細胞發炎反應之機轉探討 | zh_TW |
dc.title | The anti-inflammatory mechanisms of pcpc07f2 on lipopolysaccharide-stimulated phagocytes | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧哲明,楊春茂,顏茂雄 | |
dc.subject.keyword | 發炎反應,細胞激素,巨噬細胞, | zh_TW |
dc.subject.keyword | inflammation,cytokine,macrophage, | en |
dc.relation.page | 77 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-29 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥理學研究所 | zh_TW |
顯示於系所單位: | 藥理學科所 |
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