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研究生:廖林瑜
研究生(外文):Lin-Yu Liao
論文名稱:探討Src在H2O2刺激巨噬細胞分泌TNF-α的角色
論文名稱(外文):The role of Src in TNF-α secretion in H2O2-stimulated macrophages
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:基礎醫學研究所碩士班
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:2015
畢業學年度:103
語文別:中文
論文頁數:48
中文關鍵詞:巨噬細胞Src腫瘤壞死因子過氧化氫
外文關鍵詞:MacrophagesSrcTNF-αH2O2
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免疫系統有分為物理性免疫、先天免疫和後天性免疫,屬於先天性免疫的巨噬細胞是很重要的。當人類感染病原體,巨噬細胞會被激活並釋放大量的活性氧化物(ROS),在免疫系統中執行各種細胞功能。ROS可刺激細胞,使DNA和蛋白質產生氧化損傷,並導致細胞衰老、發炎和細胞凋亡。巨噬細胞會分泌促發炎細胞因子,如腫瘤壞死因子(TNF-α);感染或損傷的條件下,TNF-α會分泌。在這項研究中,我們使用過氧化氫(H2O2)作為活性氧化物。H2O2處理的RAW264.7巨噬細胞,會使Src的蛋白質表現量增加、增加Tyrosine phosphorylation (p-Tyr)和TNF-α分泌。上述變化大小與H2O2的使用劑量呈正相關。此外,抑制劑, PP2抑制了H2O2引發的TNF-α分泌。由於Lyn、Fgr、Hck(Src家族成員)蛋白表現量未因H2O2而有顯著改變。故Lyn、Fgr與Hck可能不是參與在H2O2所引發的信號傳導。在RAW 264.7 細胞用RNA interference的技術減少Src 表現量,會減弱H2O2誘發的細胞分泌TNF-α;而回復Src 表現,則可恢復細胞分泌TNF-α。從上述結果,我們總結H2O2刺激後的巨噬細胞,會因Src的表現量的提高和活性增加,分泌TNF-α。

Immune systems include anatomical and physiological barriers, innate immunity and adaptive immunity. Macrophages are important for immunity. When humans are infected by pathogens, macrophages are activated and release large amounts of reactive oxygen species (ROS) to perform a variety of cellular functions. ROS can stimulate cellular respiration, provoke oxidative damage in DNA and proteins, cause cell senescence, inflammation, and apoptosis. Macrophages are a major source of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), which are expressed during conditions of inflammation, infection, or injury. In this study, we use hydrogen peroxide (H2O2) as the source of ROS. We demonstrate the upregulation of Src and increased total tyrosine phosphorylation, and TNF-α secretion in H2O2-treated RAW264.7 macrophages. This H2O2-mediated TNF-α secretion was dose-dependent. Interestingly, an inhibitor for Src family kinases, PP2 can block this effect. Given that no significant alteration of the expression of Lyn, Fgr, Hck (the myeloid Src relatives) was observed, therefore, they might not be the major players in H2O2-mediated signaling pathway. Remarkably, silencing of Src expression by RNA interference reduced H2O2-evoked TNF-α secretion in RAW 264.7 macrophages and restored Src expression could rescue this defect. Based on these results, we concluded that through the induced expression and activation of Src, H2O2 treatment led to TNF-α secretion.

中文摘要 (Abstract in Chinese) I
英文摘要 (Abstract in English) II
致謝 III
第一章 前言 (Introduction) 1
第一節 免疫系統 (immune system)與巨噬細胞 (macrophage) 2
第二節 Src kinase 5
第三節Tumor necrosis factor-α (TNF-α) 8
第四節Reactive oxygen species( ROS) 9
第二章 研究動機 (Motive) 11
第三章 實驗材料及方法(Materials and Methods) 13
第一節 實驗材料 14
一、細胞: 14
二、試劑: 15
三、抗體: 16
第二節 實驗方法 17
一、細胞培養 (cell culture) : 17
二、收集細胞 lysates: 19
三、蛋白濃度測定: 20
四、蛋白質電泳(SDS-PAGE): 21
五、TNF-α測定 : 22
第四章 結果 (Results) 25
一、H2O2會增加巨噬細胞株RAW264.7釋出TNF-α 26
二、Src family kinases參與在H2O2刺激的巨噬細胞RAW264.7分泌TNF-α。 27
三、巨噬細胞RAW264.7在H2O2的刺激下會隨著濃度而增加Src的表現量與TNF-α。 28
四、巨噬細胞RAW264.7在H2O2的刺激下會隨著時間而增加Src的表現量與分泌TNF-α。 29
五、NAC抑制LPS刺激後的巨噬細胞RAW264.7分泌TNF-α的產生 30
六、Src參與在H2O2所誘發的巨噬細胞分泌TNF-α 31
第五章 討論 (Discussion) 32
第六章 參考文獻 (Reference) 36
第七章 圖表 (Figure) 42
Figure 1. H2O2 induce total phospotyrosine content in macrophages. 43
Figure 2. PP2 suppressed the H2O2-induced TNF-α and Src in RAW264.7 cells. 44
Figure 3. Effect of H2O2 on the production of TNF-α and Src in RAW264.7 cells. 45
Figure 4. Time course of H2O2-induced TNF-α and Src production in RAW 264.7 cells. 46
Figure 5. ROS inhibitor, NAC, inhibited LPS-induced Src protein level. 47
Figure 6. H2O2-mediated macrophages secretion TNF-α was inhibited by src-specific siRNA but ectopic Src could reverse this phenomenon. 48



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