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研究生:黃春霖
研究生(外文):Chun-Lin Huang
論文名稱:iNOS及Src於LPS誘發IL-6的角色探討
論文名稱(外文):The Role of Inducible Nitric Oxide Synthase and Src for LPS-Stimulated Interleukin-6 Secretion
指導教授:馬明琪
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:50
中文關鍵詞:發炎反應iNOSSrcMAPK巨噬細胞
外文關鍵詞:InflammationiNOSSrcMAPKmacrophages
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無論就先天性免疫或後天性免疫而言,cytokines中interleukin-6的多功能性扮演著一個很重要的角色。IL-6除了在感染初期做為急性發炎反應的媒介之外,在接下來的適應性免疫中,IL-6也參與了B細胞產生抗體的過程、調節regulatory T細胞對抗微生物感染等。在我們的研究中,我們觀察到了,在利用LPS剌激macrophages時,inducible nitric oxide synthase (iNOS) 會被誘導出來,並且有IL-6的釋出。這使人聯想到iNOS的產物 -- NO這個次級訊息傳遞分子可能會對於IL-6的產生有著影響。另外,Src已經被證實了對於在LPS的剌激之下macrophages的活化有著調節的作用。我們使用了抑制iNOS活性的藥物以及iNOS knockout mice的實驗結果皆顯示出iNOS對於IL-6的產出有著很大的影響;而在使用Src的抑制劑後,發現Src也對IL-6的釋出有著調節的作用。接著,在加入了NO donor -- SNAP及cGMP homolog -- 8-br-cGMP之後,都能使wild type和iNOS null mice的腹腔巨噬細胞誘發出IL-6。這些發現指出做為NO/cGMP pathway的下游標的,Src對於LPS剌激之下macrophages產生IL-6是不可或缺的。
Interleukin-6 (IL-6) is a multifunctional cytokine that plays a pivotal role in both innate and adaptive immunity. As a predominant mediator of the acute phase response triggered by infection and inflammation, IL-6 also participates in the subsequent development of acquired immunity against pathogens such as stimulation of antibody production by B cells and regulation of the response of regulatory T cells to microbial infection. In this study, we observed concomitant induction of inducible nitric oxide synthase (iNOS) and generation of IL-6 in LPS-treated macrophages. This raised a possibility that NO, a well-known second messenger produced by iNOS, might contribute to IL-6 production. In addition, Src is also a well-studied tyrosine kinase and is proved to be an important regulator in LPS-induced macrophage migration. Pharmacological blockade or knockout of iNOS reduced LPS-induced IL-6 secretion. Inhibition of Src kinase activity caused the reduction of LPS-stimulated IL-6 expression. Interestingly, SNAP (a NO donor) or 8-br-cGMP (a cGMP analogue) provoked the production of IL-6 in both wild type and iNOS null macrophages. These findings indicate that iNOS and Src are indispensable in LPS-elicited IL-6 production. These finds indicated that as a downstream target of NO/cGMP pathway, Src plays a critical role in LPS-triggered IL-6 generation.
致謝……………………………………………………………………………………………Ⅲ
中文摘要 (Abstract in Chinese)……………………………………………………………………Ⅳ
英文摘要 (Abstract in English)…………………………………………………………………Ⅴ
第一章 序論 (Introduction)………………………………………………………………………..1
1) 免疫系統及巨噬細胞的活化……………….……………………………………………….1
2) Cytokines 及IL-6.…………………………………………………………………………...2
3) Nitric Oxide 及 iNOS…….…………………………………………………………………4
4) Src Family Kinases 及 Src 參與巨噬細胞的活化………………………………...………6
5) MAPK Pathway 與 ERK Kinase 及其下游途徑…….…………………………………8
第二章 實驗材料與方法 (Materials and Methods)……………………………………………….10
第一節 實驗材料…………………………………………………………………………………..10
1) 動物…………………………………………………………………………………………..10
2) 細胞…………………………………………………………………………………………..10
3) 試劑…………………………………………………………………………………………..10
4) 實驗儀器與耗材……………………………………………………………………………..13
第二節 實驗方法…………………………………………………………………………………..14
1) 細胞培養……………………………………………………………………………………..14
2) 收集Cell lysates……………………………………………………………………………..17
3) 蛋白質濃度測定……………………………………………………………………………..18
4) 蛋白質電泳 (SDS-PAGE)………………………………………………………………….19
5) Western blot analysis……………………………………………………………………….22
6) IL-6測定……………………………………………………………………………………..24
第三章 結果 (Results)………………………………………………………………………………27
1) LPS可以誘導RAW264.7 macrophage 的 iNOS,Src及IL-6表現增加……….…….27
2) AG及ODQ可以抑制在LPS剌激之下 macrophage 所產生的Src 與IL-6…………27
3) PP2可以抑制RAW264.7 cell line 產生IL-6………………………………………………28
4) LPS無法誘導iNOS knockout mice peritoneal macrophage產生IL-6…………………28
5) SNAP及8-br-cGMP可以誘導iNOS knockout mice PEM產生IL-6…………………..29
6) PD98059可以有效地抑制巨噬細胞分泌IL-6……………………………………………..29
7) AG能有效抑制LPS誘導pERK的表現………………………………………………….30
第四章 討論 (Discussion)…………………………………………………………………………...31
參考文獻 (References)………………………………………………………………………………..35
圖表 (Figures)…………………………………………………………………………………………41
Figure.1…………………………………………………………………………………………..41
Figure.2…………………………………………………………………………………………..42
Figure.3…………………………………………………………………………………………..44
Figure.4…………………………………………………………………………………………..45
Figure.5…………………………………………………………………………………………..46
Figure.6…………………………………………………………………………………………..47
Figure.7…………………………………………………………………………………………..48
Figure.8…………………………………………………………………………………………..49
附圖、一………………………………………………………………………………………….50
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