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研究生:謝銘育
研究生(外文):Ming-Yu Hsieh
論文名稱:LPS、PGN刺激巨噬細胞產生TNFα是透過iNOS所調控的sustainedERKactivation
論文名稱(外文):Sustained ERK activation mediated by iNOS is required for LPS- and PGN-induced TNFα
指導教授:馬明琪
指導教授(外文):Ming-Chei Maa
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:68
中文關鍵詞:巨噬細胞發炎
外文關鍵詞:LPSPGNiNOSTNFα
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Toll-like receptors (TLRs)對於巨噬細胞來說扮演著重要的角色,其主要功能有接受外來的訊息以及誘發發炎相關的激素,如tumor necrosis factor-alpha (TNFa)。TLR4可辨識lipopolysaccharide (LPS)、革蘭氏陰性菌的細胞壁成分之一,TLR2則被peptidoglycan (PGN)活化,革蘭氏陽性菌細胞壁的主要成分之一。在先前的研究中認為由LPS與PGN刺激巨噬細胞所產生的iNOS與TNFa並行互不干擾的,但iNOS有可能參與在調控TNFa的路徑中。透過藥物的抑制以及基因剃除iNOS的表現的確使LPS與PGN刺激巨噬細胞產生TNFa的釋放量降低,另一方面,SNAP (a NO donor) or 8-br-cGMP (a cGMP analogue)皆可以促使WT或是iNOS null mice巨噬細胞的TNFa表現量量增加。(1) PP2可以抑制LPS或PGN刺激巨噬細胞產生TNFa(2) NO可使Src的表現量增加,(3) TNFa的轉錄是需要ERK,上述的原因,存在著一個可能性, NO促進Src的增加是與LPS、PGN刺激巨噬細胞所產生的TNFa表現量有關。由實驗結果可知,PD98059 (MEK的抑制劑) 以及Src siRNA的細胞皆可有效的抑制LPS、PGN、SNAP、8-br-cGMP刺激巨噬細胞所造成的ERK activation以及TNFa的釋放量。基於實驗結果指出iNOS、ERK activation與TNFa存在著重要的關聯性,可能是LPS、PGN、SNAP、8-br-cGMP透過iNOS活化Src和ERK去調控TNFa的表現量
Toll-like receptors (TLRs) are important for macrophages to recognize foreign insults and induce the secretion of proinflammatory cytokines such as tumor necrosis factor-alpha (TNFa). While TLR4 can recognize lipopolysaccharide (LPS), a Gram-negative bacterial component; TLR2 acts as a signaling receptor for peptidoglycan (PGN), a major component of the cell wall of Gram-positive bacteria. Concomitant induction of nitric oxide synthase (iNOS) and TNFa in LPS- and PGN-stimulated macrophages raised the possibility that iNOS was involved in TNFa generation. Indeed, pharmacological blockade or knockout of iNOS reduced LPS- and PGN-mediated TNFa secretion. Meanwhile, SNAP (a NO donor) or 8-br-cGMP (a cGMP analogue) induced the production of TNFa in both wild type and iNOS null macrophages. Since (1) LPS- and PGN-increased TNFa generation was PP2-sensitive, (2) NO could upregulate the expression of Src, and (3) ERK was required for transcription of tnfa?ngene; therefore, one might wonder NO-mediated Src enhancement was involved in generation of TNFa in macrophages exposed to LPS and PGN. Indeed, LPS-, PGN-, SNAP-, and 8-br-cGMP-mediated activation of ERK as well as induction of TNFa were greatly suppressed in macrophages treated with PD98059 (the MEK inhibitor) and attenuation of Src by src specific siRNA reduced LPS- and PGN-evoked ERK activation and TNFa release in Raw264.7 macrophages and reintroduction of siRNA-resistant Src could reverse these events. With these results, the strong association between ERK activation and TNFa secretion in macrophages devoid of iNOS exposed to LPS, PGN, SNAP and 8-br-cGMP indicated that via activation of Src and ERK, iNOS was required for LPS-and PGN-elicited TNFa generation.
目錄 I
致謝 IV
ABSTRACT VI
中文摘要 VII
壹、 序論 (INTRODUCTION) 1
第一節 免疫系統 (IMMUNE SYSTEM) 與巨噬細胞 (MACROPHAGES) 2
第二節 內毒素 (ENDOTOXIN) 2
第三節 TOLL-LIKE RECEPTOR (TLR) 4
第四節 INOS (INDUCIBLE NITRIC OXIDE SYNTHASE) 5
第五節 SRC KINASE 6
第六節 活化蛋白質激酶 (MITOGEN-ACTIVATED PROTEIN KINASE,MAPK KINASE) ………………………………………………………………………….8
貳、 實驗材料及方法 10
第一節 實驗設計流程 11
第二節 實驗材料 12
第三節 實驗方法 16
參、 結果 (RESULTS) 32
一、 AG及ODQ皆可同步抑制PGN所產生的 TNFa,且SNAP及8-BR-CGMP處理RAW 264.7巨噬細胞,皆可增加TNFa的釋放。 33
二、 SNAP與8-BR-CGMP可誘導INOS KNOCKOUT MICE PERITONEAL MACROPHAGE 產生TNFa 34
三、 AG及ODQ皆能有效抑制PGN誘導ERK的活化 35
四、 LPS與PGN誘導ERK的活化有兩個不同的階段 36
五、 LPS與PGN所調控的SUSTAINED ERK 的活性是會受AG的影響 36
六、 INOS為PGN-MEDIATED SUSTAINED ERK ACTIVATION所需 37
七、 SRC FAMILY KINASES參與在LPS與PGN所誘導的SUSTAINED ERK ACTIVATION。 38
八、 SRC參與在LPS、PGN與SNAP所誘導巨噬細胞的SUSTAINED ERK ACTIVATION及TNFa PRODUCTION。 39
肆、 討論 ( DISCUSSION ) 41
伍、 參考文獻(REFERENCE) 46

圖表目錄
陸、 圖表(FIGURE) 52
FIGURE 1. NO/CGMP IS INVOLVED IN PGN-INDUCED TNFa SECRETION. 53
FIGURE 2. INOS IS REQUIRED FOR TNFa SECRETION MEDIATED BY LPS AND PGN. 55
FIGURE 3. PGN-INDUCED ERK ACTIVATION IS INHIBITOR BY AG OR ODQ. 56
FIGURE 4. SUSTAINED ERK ACTIVATION MEDIATED BY LPS AND PGN. 57
FIGURE 5. LPS- AND PGN-MEDIATED SUSTAINED ERK ACTIVATION WAS AG-SENSITIVE. 58
FIGURE 6. INOS IS INVOLVED IN PGN-MEDIATED ERK ACTIVATION. 59
FIGURE 7. INVOLVEMENT OF SRC FAMILY KINASES IN THE LATE STAGE OF LPS- AND PGN-INDUCED TNFa SECRETION AND ERK ACTIVATION. 60
FIGURE 8. LPS- AND PGN-MEDIATED TNFa SECRETION WAS INHIBITED BY SRC-SPECIFIC SIRNA. 61
FIGURE 9. LPS- AND PGN-MEDIATED TNFa SECRETION WAS INHIBITED BY SRC-SPECIFIC SIRNA, WHICH COULD BE REVERSED BY ECTOPIC SRC. 62
FIGURE 10. SNAP MEDIATED TNFa SECRETION WAS INHIBITED BY SRC-SPECIFIC SIRNA BUT ECTOPIC SRC COULD REVERSE THIS PHENOMENON. 63
FIGURE 11. THE PROPOSED NO/SGC/SRC/ERK SIGNALING PATHWAY FOR TNFa SECRETION IN LPS- AND PGN- STIMULATED MACROPHAGES. 64
附圖一、STRUCTURE OF LIPOPOLYSACCHARIDE AND PEPTIDOGLYCAN. 65
附圖二、LPS-INDUCED TNFa TRANSCRIPT IS SENSITIVE TO INHIBITORS OF INOS AND SGC. 66
附圖三、MEK INHIBITOR PD98059 SUPPRESSED THE SNAP- AND 8-BR-CGMP-INDUCED TNFa PRODUCTION. 67
附圖四、LPS-MEDIATED ERK ACTIVATION WAS ABROGATED IN INOS NULL MACROPHAGES, WHICH COULD BE RESTORED BY SNAP AND 8-BR-CGMP. 68
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