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研究生:楊佳靜
研究生(外文):Jia-Jing Yang
論文名稱:Pleurocidin調節LPS引起之發炎反應的研究
論文名稱(外文):Study on the regulation of Pleurocidin in LPS-induced inflammatory responses
指導教授:楊忠達
指導教授(外文):Chung-Da Yang
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:動物疫苗科技研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:75
中文關鍵詞:脂多醣抗菌胜肽Pleurocidin發炎反應
外文關鍵詞:LipopolysaccharideAntimicrobial peptidesPleurocidinInflammatory responses
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脂多醣 (Lipopolysaccharide; LPS)為革蘭氏陰性菌細胞膜上主要的成份之一,亦是細菌入侵的標記。當微生物入侵並釋放大量的LPS後,會刺激免疫細胞分泌大量的促發炎細胞激素,如TNF-,並使宿主產生過度發炎反應,甚至產生敗血症。抗菌胜肽 (Antimicrobial peptides; AMPs)是種帶正電的陽離子胜肽,廣泛存在於植物、昆蟲、魚類、兩棲類及哺乳動物中,且在宿主的先天防禦機制上被認為扮演重要的角色,同時具有抗微生物的活性,可對抗細菌、真菌,甚至是病毒。Pleurocidin (Ple)是由25個胺基酸所合成的抗菌胜肽,具有-helical的結構,最初從美洲鰈 (Pleuronectes americanus)皮膚上所分泌的黏液而來,可經由破壞細胞膜結構來抑制微生物生長。近年,已有許多文獻指出AMPs具有中和LPS作用,以降低宿主發生過度發炎反應的機率,因此本研究以小鼠模式來觀察Ple對於LPS誘導所產生的發炎反應是否具有中和作用,並探討其中機制。研究結果發現,經LPS (80 pg/ml)及Ple (80 g/ml)共同刺激4小時後,小鼠巨噬細胞所分泌的TNF-有明顯的降低。此外,由小鼠動物試驗結果顯示,以LPS 0.1 g/mouse與Ple 0.1 mg/mouse腹腔注射後,能顯著性降低小鼠血清中TNF-含量,因此無論經由體外細胞試驗或小鼠動物實驗皆可觀察到Ple可負調節經由LPS誘導而大量表現的TNF-。除此之外,我們更進一步探討Ple與LPS之間的訊息傳遞關係,由研究結果顯示,Ple的負調節作用可能與MAPK (Mitogen-activated protein kinases)路徑的下游分子ERK (Extracellular signal-regulated kinase)相關,因此抗菌胜肽Ple能經由MAPK/ERK路徑抑制經由LPS誘導所大量分泌的TNF-,進而降低發生過度發炎反應的機率。
關鍵字:脂多醣、抗菌胜肽、Pleurocidin、發炎反應
Lipopolysaccharide (LPS) is the major molecular component of the outer membrane of Gram-negative bacteria and it also recognized by the immune system as a marker for the detection of bacterial pathogen invasion. During bacterial invasion, released LPS stimulates immune cells and causes inflammatory responses even sepsis accompanied by secreting a large amount of pro-inflammatory cytokines such as TNF-. Antimicrobial peptides (AMPs) are cationic peptides and have been considered to play important roles in the innate defense mechanisms of most living organisms including plants, insects, fish, amphibians and mammals. They are also known to possess potent antimicrobial activity against bacteria, fungi, and even viruses. Pleurocidin (Ple) is a cationic -helical AMP derived from the skin mucous of the winter flounder (Pleuronectes americanus) and can inhibit the growth of a broad spectrum of microbes through membrane disruption. Many studies have suggested that AMPs have LPS-neutralizing activity to decrease TNF- associated inflammatory responses in host. In our study, we used mouse model to examine whether Ple could neutralize the LPS-induced inflammatory response. We found that TNF- secretion was significantly reduced in mouse macrophage cell line RAW 264.7 after co-stimulated with LPS (80 pg/ml) and Ple (80 g/ml) for 4 hours. In addition, after intraperitoneal injection (IP) with LPS 0.1g/mouse and Ple 0.1 mg/mouse, TNF- secretion was significantly decreased in mouse serum. Therefore, Ple showed the negative regulation of LPS-induced TNF- secretion in vitro and vivo. We further studied the signal transduction of the negative regulation in Raw 264.7 cells. We demonstrated that the negative regulation was induced via ERK (Extracellular signal-regulated kinase) of MAPK (Mitogen-activated protein kinases) signal pathway. Taken together, Ple can inhibit LPS-induced TNF- secretion through the ERK of MAPK signal pathway and further suppress excessive inflammatory response.
Keywords: Lipopolysaccharide, Antimicrobial peptides, Pleurocidin, Inflammatory responses
摘要 ....................................................I
Abstract.............................................. III
誌謝 .................................................. V
目錄 ................................................... VI
圖表目錄 ................................................ X
壹、緒言................................................. 1
貳、文獻回顧 ............................................. 3
一、脂多醣(Lipopolysaccharide; LPS)....................... 3
(一) 組成及特性 .......................................... 3
(二) 作用途徑 ............................................ 3
(三) 訊息活化路徑 ........................................ 4
1.NF-B 路徑 (NF-B pathway)............................. 4
2.Mitogen-activated protein kinases 路徑 (MAPK pathway). 5
(四) 發炎反應 (Inflammatory responses) ................ 6
(五) 敗血症 (Sepsis) ................................... 7
二、抗菌胜肽 (Antimicrobial peptides; AMPs) .............. 7
(一) 歷史................................................. 7
(二) 來源及特性 ........................................... 8
(三) 分類................................................ 9
(四) 殺菌機制 ............................................ 10
(五) 膜選擇性 (Membrane selectivity)..................... 10
(六) 免疫調節 .......................................... 11
(七) 應用................................................ 12
三、Pleurocidin (Ple) ................................... 12
四、研究目的 ............................................. 13
參、材料方法 ............................................. 14
一、抗菌胜肽 Ple ........................................ 14
二、體外試驗:小鼠巨噬細胞株 Raw 264.7 ..................... 14
(一) 細胞株培養 .......................................... 14
1.細胞株解凍............................................. 14
2.細胞株繼代培養 ......................................... 15
3.細胞株冷凍保存 ........................................ 15
(二) 細胞存活率分析 (Cell Survival Rate Assay)........... 15
1.細胞株分盤培養 ....................................... 15
2.Ple 刺激小鼠巨噬細胞 ................................... 16
3.測定細胞存活率 (MTT assay) .......................... 16
(三) 測定小鼠巨噬細胞之 TNF- 濃度......................... 16
1.以不同濃度 LPS 刺激小鼠巨噬細胞 Raw 264.7 ............. 16
2.以 Ple 及 LPS 刺激小鼠巨噬細胞 Raw 264.7.................. 17
3.以 ERK inhibitor-U0126 刺激小鼠巨噬細胞 Raw 264.7........ 17
4.酵 素 連 結 免 疫 吸 附 法 (Enzyme-linked Immunosorbent
Assay; ELISA).......................................... 18
三、小鼠活體試驗........................................ 19
四、探討訊息傳遞途徑...................................... 19
(一) 以 Ple 及 LPS 刺激小鼠巨噬細胞 Raw 264.7 ............ 19
(二) 以 ERK inhibitor-U0126 刺激小鼠巨噬細胞 Raw 264.7..... 19
(三) 萃取細胞蛋白質 .................................... 20
(四) 十二烷基硫酸鈉聚丙烯醯胺膠體電泳 (Sodium Dodecyl
Sulfate Polyacrylamide Gel Electrophoresis SDS-PAGE) .. 20
(五) 西方墨點法 (Western blotting)...................... 21
1.蛋白質轉漬 (Transfer) ............................... 21
2.抗體雜交 (Hybridization)............................. 21
3.壓片 .............................................. 22
4.抗體再作用........................................... 22
肆、結果............................................. 23
一、體外試驗:小鼠巨噬細胞株 Raw 264.7 ................... 23
(一) 小鼠巨噬細胞株 Raw 264.7............................. 23
(二) 細胞存活率分析 (Cell Survival Rate Assay)......... 23
(三) 測定小鼠巨噬細胞 TNF- 濃度......................... 23
二、小鼠活體試驗......................................... 24
三、探討訊息傳遞途徑..................................... 25
伍、討論.......................................... 39
參考文獻 ........................................... 45
附錄 ............................................. 58
作者簡介 .............................................. 63

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