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研究生:杜宣諭
研究生(外文):Tu, Hsuan-Yu
論文名稱:衍生自CXCL8的胜肽在巨噬細胞中的抗發炎反應
論文名稱(外文):Anti-inflammatory effect of a peptide derived from CXCL8 in macrophages
指導教授:江信仲
指導教授(外文):Jiang, Shinn-Jong
口試委員:許豪仁江信仲林進裕
口試委員(外文):Hsu, Hao-JenJiang, Shinn-JongLin, Chin-Yu
口試日期:2018-05-11
學位類別:碩士
校院名稱:慈濟大學
系所名稱:生物化學碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:60
中文關鍵詞:介白素8
外文關鍵詞:CXCL8
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細胞介素8藉由激活CXCR1/2而在調節發炎反應及乳癌形成中扮演重要角色。在我們先前的研究中,利用電腦模擬合成衍生自CXCL8結合位的peptide RF16,用於抑制CXCL8及TNF-誘導單核球細胞黏附和遷移。然而,peptide RF16對於THP-1細胞抗發炎的影響仍不清楚。我們研究的結果顯示,在THP-1細胞中,RF16確實降低了TNF-、CXCL8、IL-6及IL-1等促發炎因子的mRNA表現。而且也降低促發炎因子所誘導IL-6、IL-1及TNF-蛋白釋放量。此外,我們發現RF16能夠抑制TNF-及CXCL8誘導的ROS產生,進而抑制發炎反應。在LPS誘導的急性敗血症模式老鼠中,RF16能夠減少血液中白血球細胞數,也因此減少IL-6及IL-1蛋白的釋放量。此外,我們發現RF16經由NF-B、MAPK及PI3K路徑達到抑制發炎反應產生。
總體而言,在體外實驗中,RF16經由NF-B、MAPK及PI3K路徑來抑制TNF-及CXCL8誘導的發炎反應,在動物實驗則看到RF16降低了LPS所誘導的發炎反應。

Interleukin 8 (CXCL-8, IL-8) is crucial for the regulation of inflammation and breast cancer formation via the activation of its cognate receptor CXCR1/2. In our previous study, RF16, a peptide derived from CXCL8 binding region, was synthesized for the inhibiting CXCL8- induced and LPS-induced monocytes adhesion and transmigration. However, the effects of RF16 on THP-1 cells in regard to anti-inflammation were still unknown. Our results shown here indicate that the mRNA expression levels of the inflammatory cytokines TNF-, CXCL8, IL-6 and IL-1 were reduced by RF16 in THP-1 cells. The reductions in pro-inflammatory cytokines correlated to the reduced secretions of IL-6 and IL-1 proteins. In addition, we found that RF16 was able to inhibit TNF-/CXCL8 induced ROS generation, thereby suppressed the inflammatory response. Moreover, in LPS induced sepsis animal model, RF16 was able to reduce the number of white blood cells in peripheral blood, and consequently reduced the secretions of IL-6 and IL-1 proteins. The mainly mechanisms were through NF-kB/MAPK/PI3K signaling pathways to inhibit inflammation. Overall, our results demonstrated that RF16 has the ability to suppress TNF-, CXCL8 and LPS -induced pro-inflammatory cytokine expressions and secretions in both in vitro and in vivo.
目錄
Abstract 1
中文摘要 2
緒論 3
1. 發炎反應之簡介 3
2. 單核球細胞 (Monocyte) 及巨噬細胞 (Macrophage) 與發炎反應關係 3
3. 細胞激素 (Cytokines) 與趨化因子 (Chemokines) 之介紹 5
4. TNF-a與CXCL-8發炎相關訊息傳遞路徑 6
5. 胜肽藥物應用及發展 7
6. 研究動機 8
實驗材料與方法 10
1. 儀器 10
2. 藥品與試劑 11
3. 人類單核球細胞 (Human monocytic cell line, THP-1) 培養 16
4. 細胞增殖實驗 (WST-1 assay) 17
5. 氧化壓力的檢測 (Reactive Oxygen Species) 17
6. 氧化低密度脂蛋白吸收 (oxided Low Density Lipid Uptake) 18
7. 定量反轉錄聚合酶連鎖反應 (Quantitative Reverse Transcription PCR;qRT-PCR) 19
8. Enzyme-linked immunosorbent assay (ELISA) 22
9. 細胞全蛋白之萃取 (Total protein) 24
10. 細胞核蛋白之萃取 (Nuclear protein) 24
11. 蛋白質濃度檢測及SDS-PAGE loading sample製備 25
12. 西方墨點法與SDS-PAGE 26
13. 敗血症老鼠模式 29
14. 分析方法 29
結果 30
1. 測試合成胜肽RF16及CF25對於THP-1細胞毒性影響 30
2. 合成胜肽RF16降低TNF-a及IL-8誘導促發炎因子mRNA表現 30
3. 合成胜肽RF16降低TNF-a及IL-8誘導促發炎因子的釋放 30
4. 合成胜肽RF16降低TNF-a及IL-8誘導ROS的生成 31
5. 合成胜肽RF16降低TNF-a及IL-8誘導氧化低密度脂蛋白的吞噬 31
6. 合成胜肽RF16影響TNF-a及IL-8誘導THP-1細胞中的NF-kB路徑 32
7. 合成胜肽RF16影響TNF-a誘導THP-1細胞中的MAPK/P38/JNK路徑 32
8. 合成胜肽RF16影像IL-8誘導THP-1細胞中的PI3K/Akt/ERK路徑 33
9. 合成胜肽RF16抑制模擬急性敗血症動物模式中的發炎反應 33
結論與討論 34
圖表 38
圖 1. RF16及CF25對人類單核球細胞生長之影響 38
圖2. RF16在THP-1細胞中抑制TNF-a誘導促發炎因子mRNA表現 39
圖3. RF16在THP-1細胞中抑制IL-8誘導促發炎因子mRNA表現 40
圖4. RF16在THP-1細胞中抑制TNF-a誘導促發炎因子的釋放 41
圖5. RF16在THP-1細胞中抑制IL-8誘導促發炎因子的釋放 42
圖6. RF16在THP-1細胞中抑制TNF-a誘導ROS的生成 44
圖7. RF16在THP-1細胞中抑制IL-8誘導ROS的生成 46
圖8. RF16在THP-1細胞中抑制TNF-a及IL-8誘導氧化低密度脂蛋白的吞噬 47
圖9. RF16減少THP-1細胞中TNF-a及IL-8誘導的核內P65蛋白表現量 48
圖10. RF16降低THP-1細胞中TNF-a誘導的MAPK/P38/JNK訊號蛋白表現量 49
圖11. RF16降低THP-1細胞中IL-8誘導的PI3K/Akt/ERK訊號蛋白表現量 50
圖12. RF16降低LPS誘導模擬急性敗血症模式中的白血球數及促發炎因子的釋放 51
附錄 52
1. Real time PCR primer (Human) 52
參考文獻 53


參考文獻
1.Holmes WE, Lee J, Kuang WJ, Rice GC, & Wood WI (1991) Structure and functional expression of a human interleukin-8 receptor. Science 253(5025):1278-1280.
2.Fernandez EJ & Lolis E (2002) Structure, function, and inhibition of chemokines. Annu Rev Pharmacol Toxicol 42:469-499.
3.Luster AD (2002) The role of chemokines in linking innate and adaptive immunity. Curr Opin Immunol 14(1):129-135.
4.Ulbrich H, Eriksson EE, & Lindbom L (2003) Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol Sci 24(12):640-647.
5.Moser B, Wolf M, Walz A, & Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends Immunol 25(2):75-84.
6.Shacter E & Weitzman SA (2002) Chronic inflammation and cancer. Oncology (Williston Park) 16(2):217-226, 229; discussion 230-212.
7.Libby P, Ridker PM, & Maseri A (2002) Inflammation and atherosclerosis. Circulation 105(9):1135-1143.
8.Coussens LM & Werb Z (2002) Inflammation and cancer. Nature 420(6917):860-867.
9.Wellen KE & Hotamisligil GS (2005) Inflammation, stress, and diabetes. J Clin Invest 115(5):1111-1119.
10.Yang J, Zhang L, Yu C, Yang XF, & Wang H (2014) Monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases. Biomark Res 2(1):1.
11.Mansour SC, Pena OM, & Hancock RE (2014) Host defense peptides: front-line immunomodulators. Trends Immunol 35(9):443-450.
12.Mosser DM & Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8(12):958-969.
13.Rios FJ, et al. (2013) Oxidized LDL induces alternative macrophage phenotype through activation of CD36 and PAFR. Mediators Inflamm 2013:198193.
14.Baggiolini M, Moser B, & Clark-Lewis I (1994) Interleukin-8 and related chemotactic cytokines. The Giles Filley Lecture. Chest 105(3 Suppl):95S-98S.
15.Apostolakis S, Vogiatzi K, Amanatidou V, & Spandidos DA (2009) Interleukin 8 and cardiovascular disease. Cardiovasc Res 84(3):353-360.
16.Wang N, et al. (1996) Interleukin 8 is induced by cholesterol loading of macrophages and expressed by macrophage foam cells in human atheroma. J Biol Chem 271(15):8837-8842.
17.Tracey D, Klareskog L, Sasso EH, Salfeld JG, & Tak PP (2008) Tumor necrosis factor antagonist mechanisms of action: a comprehensive review. Pharmacol Ther 117(2):244-279.
18.Gupta S (2002) Tumor necrosis factor-alpha-induced apoptosis in T cells from aged humans: a role of TNFR-I and downstream signaling molecules. Exp Gerontol 37(2-3):293-299.
19.Lobito AA, Gabriel TL, Medema JP, & Kimberley FC (2011) Disease causing mutations in the TNF and TNFR superfamilies: Focus on molecular mechanisms driving disease. Trends Mol Med 17(9):494-505.
20.Baggiolini M (2015) CXCL8 - The First Chemokine. Front Immunol 6:285.
21.Yoshimura T (2015) Discovery of IL-8/CXCL8 (The Story from Frederick). Front Immunol 6:278.
22.Godessart N & Kunkel SL (2001) Chemokines in autoimmune disease. Curr Opin Immunol 13(6):670-675.
23.Ali S & Lazennec G (2007) Chemokines: novel targets for breast cancer metastasis. Cancer Metastasis Rev 26(3-4):401-420.
24.Theiss AL, Simmons JG, Jobin C, & Lund PK (2005) Tumor necrosis factor (TNF) alpha increases collagen accumulation and proliferation in intestinal myofibroblasts via TNF receptor 2. J Biol Chem 280(43):36099-36109.
25.Chen G & Goeddel DV (2002) TNF-R1 signaling: a beautiful pathway. Science 296(5573):1634-1635.
26.Kant S, et al. (2011) TNF-stimulated MAP kinase activation mediated by a Rho family GTPase signaling pathway. Genes Dev 25(19):2069-2078.
27.Sharma B, Singh S, Varney ML, & Singh RK (2010) Targeting CXCR1/CXCR2 receptor antagonism in malignant melanoma. Expert Opin Ther Targets 14(4):435-442.
28.Russo RC, Garcia CC, Teixeira MM, & Amaral FA (2014) The CXCL8/IL-8 chemokine family and its receptors in inflammatory diseases. Expert Rev Clin Immunol 10(5):593-619.
29.Cheng GZ, et al. (2008) Advances of AKT pathway in human oncogenesis and as a target for anti-cancer drug discovery. Curr Cancer Drug Targets 8(1):2-6.
30.Knall C, Worthen GS, & Johnson GL (1997) Interleukin 8-stimulated phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Proc Natl Acad Sci U S A 94(7):3052-3057.
31.Otvos L, Jr. & Wade JD (2014) Current challenges in peptide-based drug discovery. Front Chem 2:62.
32.Fosgerau K & Hoffmann T (2015) Peptide therapeutics: current status and future directions. Drug Discov Today 20(1):122-128.
33.Jiang SJ, et al. (2015) Peptides derived from CXCL8 based on in silico analysis inhibit CXCL8 interactions with its receptor CXCR1. Sci Rep 5:18638.
34.Heidemann J, et al. (2003) Angiogenic effects of interleukin 8 (CXCL8) in human intestinal microvascular endothelial cells are mediated by CXCR2. J Biol Chem 278(10):8508-8515.
35.Narayanan R, Higgins KA, Perez JR, Coleman TA, & Rosen CA (1993) Evidence for differential functions of the p50 and p65 subunits of NF-kappa B with a cell adhesion model. Mol Cell Biol 13(6):3802-3810.
36.Kunsch C & Rosen CA (1993) NF-kappa B subunit-specific regulation of the interleukin-8 promoter. Mol Cell Biol 13(10):6137-6146.
37.Shao N, et al. (2015) Interleukin-8 upregulates integrin beta3 expression and promotes estrogen receptor-negative breast cancer cell invasion by activating the PI3K/Akt/NF-kappaB pathway. Cancer Lett 364(2):165-172.
38.Ji S, et al. (2016) Sohlh2 suppresses epithelial to mesenchymal transition in breast cancer via downregulation of IL-8. Oncotarget 7(31):49411-49424.
39.Hammond ME, et al. (1995) IL-8 induces neutrophil chemotaxis predominantly via type I IL-8 receptors. J Immunol 155(3):1428-1433.
40.Hartl D, et al. (2007) Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease. Nat Med 13(12):1423-1430.
41.Sabroe I & Whyte MK (2007) Incapacitating the immune system in cystic fibrosis. Nat Med 13(12):1417-1418.
42.Vassalli P (1992) The pathophysiology of tumor necrosis factors. Annu Rev Immunol 10:411-452.
43.Bazzoni F & Beutler B (1996) The tumor necrosis factor ligand and receptor families. N Engl J Med 334(26):1717-1725.
44.Jones AT, Gumbleton M, & Duncan R (2003) Understanding endocytic pathways and intracellular trafficking: a prerequisite for effective design of advanced drug delivery systems. Adv Drug Deliv Rev 55(11):1353-1357.
45.Shi K, et al. (2015) A pH-responsive cell-penetrating peptide-modified liposomes with active recognizing of integrin alphavbeta3 for the treatment of melanoma. J Control Release 217:138-150.
46.Giai C, et al. (2013) Shedding of tumor necrosis factor receptor 1 induced by protein A decreases tumor necrosis factor alpha availability and inflammation during systemic Staphylococcus aureus infection. Infect Immun 81(11):4200-4207.
47.Bishayi B, Nandi A, Dey R, & Adhikary R (2017) Expression of CXCR1 (IL-8 receptor A) in splenic, peritoneal macrophages and resident bone marrow cells after acute live or heat killed Staphylococcus aureus stimulation in mice. Microb Pathog 109:131-150.
48.Bishayi B, Bandyopadhyay D, Majhi A, & Adhikary R (2015) Expression of CXCR1 (interleukin-8 receptor) in murine macrophages after staphylococcus aureus infection and its possible implication on intracellular survival correlating with cytokines and bacterial anti-oxidant enzymes. Inflammation 38(2):812-827.
49.Kulbe H, Hagemann T, Szlosarek PW, Balkwill FR, & Wilson JL (2005) The inflammatory cytokine tumor necrosis factor-alpha regulates chemokine receptor expression on ovarian cancer cells. Cancer Res 65(22):10355-10362.
50.Moore RJ, et al. (1999) Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis. Nat Med 5(7):828-831.
51.Osawa Y, et al. (2002) Tumor necrosis factor alpha-induced interleukin-8 production via NF-kappaB and phosphatidylinositol 3-kinase/Akt pathways inhibits cell apoptosis in human hepatocytes. Infect Immun 70(11):6294-6301.


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