臺灣博碩士論文加值系統

嗜中性白血球是第一個到達發炎位置的免疫細胞，而第四型類鐸受體是活化嗜中性白血球的重要調節者。革蘭氏陰性菌的脂多醣為活化第四型類鐸受體的訊息傳導路徑之主要活化者，能夠誘導嗜中性白血球釋放細胞激素，促進發炎反應。多胺在許多生理的功能上扮演著重要的角色，包括細胞週期的調控、細胞的增生與免疫調節，在免疫調節方面，多胺被發現抑制促發炎細胞激素的合成，然而多胺如何調節此種免疫反應的機制仍不清楚。因此，本研究探討多胺是否透過調節第四型類鐸受體的訊息傳遞，進而抑制促發炎細胞激素表現。首先研究發現多胺能夠抑制第四型類鐸受體的mRNA和蛋白質的表現，而且多胺能夠減少脂多醣所刺激的類嗜中性白血球的IL-1b之表現。另一方面，在類嗜中性白血球上處理NF-kB抑制劑BAY-11-7082與AP-1抑制劑Curcumin發現抑制第四型類鐸受體的表現，更進一步以多胺處理發現能減少NF-kB與AP-1的表現。綜合上述結果顯示多胺是經由降低發炎前驅轉錄因子NF-kB和AP-1的表現，進而減少類嗜中性白血球上的第四型類鐸受體之表現，最後導致其訊息傳遞被抑制。

Neutrophils are the first immune cells to arrive at sites of infection. Bacterial products induce the release of proinflammatory cytokines in neutrophils. Lipopolysaccharide (LPS) is the principal outer membrane component of Gram-negative bacteria that activates Toll-like receptor 4 (TLR4) signaling pathway. TLR4 is one of the most important regulators of neutrophil activation. Polyamines play an important role in many biological functions, including cell cycle, proliferation and immunomodulation. Polyamines specifically suppress the synthesis of proinflammatory cytokines. However, how it regulates the immune responses is still unclear. Therefore, we examined whether polyamines regulated TLR4 signaling to inhibit proinflammatory cytokines . In this study, we found that polyamines inhibited TLR4 protein and mRNA expression. Moreover, polyamines repressed IL-1b expression in neutrophil-like cells to response LPS. In addition, a NF-kB inhibitor BAY-11-7082 and an AP-1 inhibitor curcumin suppressed TLR4 expression in neutrophil-like cells. We further found polyamines inhibited NF-kB and AP-1 expression in neutrophil-like cells. These results demonstrated that polyamines could inhibit TLR4 signaling through repressing proinflammatory transcription factors NF-kB and AP-1 expression in neutrophil-like cells.

中文摘要 I
英文摘要 II
目錄 III
圖目錄 V
附表目錄 VI
第一章 緒論.............. 1
1. 先天性免疫....... 1
2. 嗜中性白血球...... 2
3. 類嗜中性白血球之分化....... 5
4. 第四型類鐸受體.... 6
5. 多胺.... 8
第二章 材料與方法. 9
1. 細胞培養. 9
2. 西方墨點法分析.... 10
3. 細胞存活率分析.... 12
4. 反轉錄聚合酶連鎖反應....... 12
第三章 結果...... 15
1. 第四型類鐸受體和CD14在類嗜中性白血球上的表現... 15
2. 多胺對類嗜中性白血球存活率的影響..... 16
3. 多胺抑制類嗜中性白血球之第四型類鐸受體表現量.... 17
4. 多胺抑制類嗜中性白血球上細胞激素IL-1b表現..... 18
5. 多胺對嗜中性白血球之發炎前驅轉錄因子NF-kB 與AP-1之影響..19
第四章 討論...... 21
第五章 參考文獻... 26

圖目錄
圖 一、DMSO促進第四型類鐸受體和CD14之表現量增加 32
圖 二、多胺對類嗜中性白血球存活率的影響 34
圖 三、多胺抑制類嗜中性白血球上第四型類鐸受體的表現量.... 35
圖 四、多胺抑制類嗜中性白血球上細胞激素IL-1b的表現量.... 36
圖 五、多胺抑制類嗜中性白血球上NF-кB 與AP-1之表現...... 38

附表目錄

附表 1：Gold lysis buffer.......39
附表 2：Protein loading dye, 6X.. 40
附表 3：Running buffer, 5X....... 40
附表 4：Transfer buffer.. 41
附表 5：Wash buffer...... 41
附表 6：Phosphate buffer saline (PBS) , 10X...... 41
附表 7：cDNA合成反應溶液... 42
附表 8：PCR反應溶液....... 42
附表 9：引子序列.. 42
附表 10：DNA loading dye, 10X.... 43

Adler S, Pellizzer C, Paparella M, Hartung T, Bremer S. 2006. The effects of solvents on embryonic stem cell differentiation. Toxicol In Vitro 20(3):265-271.
Akira S, Takeda K. 2004. Toll-like receptor signalling. Nat Rev Immunol 4(7):499-511.
Breitman TR, Selonick SE, Collins SJ. 1980. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci U S A 77(5):2936-2940.
Choi YH, Park HY. 2012. Anti-inflammatory effects of spermidine in lipopolysaccharide-stimulated BV2 microglial cells. J Biomed Sci 19:31.
Collins SJ, Bodner A, Ting R, Gallo RC. 1980. Induction of morphological and functional differentiation of human promyelocytic leukemia cells (HL-60) by componuds which induce differentiation of murine leukemia cells. Int J Cancer 25(2):213-218.
Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC. 1978. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci U S A 75(5):2458-2462.
Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC. 1979. Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide. J Exp Med 149(4):969-974.
Evans MS, Reid KH, Sharp JB, Jr. 1993. Dimethylsulfoxide (DMSO) blocks conduction in peripheral nerve C fibers: a possible mechanism of analgesia. Neurosci Lett 150(2):145-148.
Foletta VC, Segal DH, Cohen DR. 1998. Transcriptional regulation in the immune system: all roads lead to AP-1. J Leukoc Biol 63(2):139-152.
Gabay C, Arend WP. 1998. Treatment of rheumatoid arthritis with IL-1 inhibitors. Springer Semin Immunopathol 20(1-2):229-246.
Gougerot-Pocidalo MA. 2012. The neutrophil and systemic inflammation. Joint Bone Spine 79(3):209-211.
Hayashi F, Means TK, Luster AD. 2003. Toll-like receptors stimulate human neutrophil function. Blood 102(7):2660-2669.
Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA. 1999. Phylogenetic perspectives in innate immunity. Science 284(5418):1313-1318.
Jacob SW, Herschler R. 1986. Pharmacology of DMSO. Cryobiology 23(1):14-27.
Kindt TJ, Goldsby RA, Osborne BA. 2011. Kuby免疫學. 高榮駿編譯, translator. 台北市: 九州圖書. 52-71 p.
Lesiak A, Sysa-Jedrzejowska A, Narbutt J, Lukamowicz J, Robak E, Wozniacka A. 2005. [Proinflammatory cytokines in inactive lupus erythematosus patients]. Przegl Lek 62(9):838-842.
Li H, Sun B. 2007. Toll-like receptor 4 in atherosclerosis. J Cell Mol Med 11(1):88-95.
Li L, Rao JN, Bass BL, Wang JY. 2001. NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 280(5):G992-G1004.
Li Y, Karlin A, Loike JD, Silverstein SC. 2002. A critical concentration of neutrophils is required for effective bacterial killing in suspension. Proc Natl Acad Sci U S A 99(12):8289-8294.
Liao YF, Hung HC, Hsu PC, Kao MC, Hour TC, Tsay GJ, Liu GY. 2008. Ornithine decarboxylase interferes with macrophage-like differentiation and matrix metalloproteinase-9 expression by tumor necrosis factor alpha via NF-kappaB. Leuk Res 32(7):1124-1140.
Liu B, Yang Y, Dai J, Medzhitov R, Freudenberg MA, Zhang PL, Li Z. 2006. TLR4 up-regulation at protein or gene level is pathogenic for lupus-like autoimmune disease. J Immunol 177(10):6880-6888.
Lovaas E, Carlin G. 1991. Spermine: an anti-oxidant and anti-inflammatory agent. Free Radic Biol Med 11(5):455-461.
Lu YC, Yeh WC, Ohashi PS. 2008. LPS/TLR4 signal transduction pathway. Cytokine 42(2):145-151.
Malcolm KC, Worthen GS. 2003. Lipopolysaccharide stimulates p38-dependent induction of antiviral genes in neutrophils independently of paracrine factors. J Biol Chem 278(18):15693-15701.
Marton LJ, Pegg AE. 1995. Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol 35:55-91.
Matsunaga N, Tsuchimori N, Matsumoto T, Ii M. 2010. TAK-242 (resatorvid), a small-molecule inhibitor of Toll-like receptor (TLR) 4 signaling, binds selectively to TLR4 and interferes with interactions between TLR4 and its adaptor molecules. Mol Pharmacol 79(1):34-41.
Medzhitov R, Preston-Hurlburt P, Janeway CA, Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388(6640):394-397.
Moinard C, Cynober L, de Bandt JP. 2005. Polyamines: metabolism and implications in human diseases. Clin Nutr 24(2):184-197.
Ngai J, Capetanaki YG, Lazarides E. 1984. Differentiation of murine erythroleukemia cells results in the rapid repression of vimentin gene expression. J Cell Biol 99(1 Pt 1):306-314.
O''Neill LA, Bryant CE, Doyle SL. 2009. Therapeutic targeting of Toll-like receptors for infectious and inflammatory diseases and cancer. Pharmacol Rev 61(2):177-197.
Parham P. 2002. 免疫系統. 黎煥耀, translator. 台北市: 偉明圖書 1-18 p.
Parker LC, Whyte MK, Dower SK, Sabroe I. 2005. The expression and roles of Toll-like receptors in the biology of the human neutrophil. J Leukoc Biol 77(6):886-892.
Patel AR, Wang JY. 1999. Polyamine depletion is associated with an increase in JunD/AP-1 activity in small intestinal crypt cells. Am J Physiol 276(2 Pt 1):G441-450.
Pegg AE. 1986. Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J 234(2):249-262.
Pegg AE, McCann PP. 1982. Polyamine metabolism and function. Am J Physiol 243(5):C212-221.
Pfeffer LM, Yang CH, Murti A, McCormack SA, Viar MJ, Ray RM, Johnson LR. 2001. Polyamine depletion induces rapid NF-kappa B activation in IEC-6 cells. J Biol Chem 276(49):45909-45913.
Poulin R, Pelletier G, Pegg AE. 1995. Induction of apoptosis by excessive polyamine accumulation in ornithine decarboxylase-overproducing L1210 cells. Biochem J 311 ( Pt 3):723-727.
Quan CP, Roux C, Pillot J, Bouvet JP. 1990. Delineation between T and B suppressive molecules from human seminal plasma: II. Spermine is the major suppressor of T-lymphocytes in vitro. Am J Reprod Immunol 22(1-2):64-69.
Quint JK, Wedzicha JA. 2007. The neutrophil in chronic obstructive pulmonary disease. J Allergy Clin Immunol 119(5):1065-1071.
Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seydel U, Di Padova F, et al. 1994. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J 8(2):217-225.
Roger T, Miconnet I, Schiesser AL, Kai H, Miyake K, Calandra T. 2005. Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression. Biochem J 387(Pt 2):355-365.
Sabroe I, Dower SK, Whyte MK. 2005a. The role of Toll-like receptors in the regulation of neutrophil migration, activation, and apoptosis. Clin Infect Dis 41 Suppl 7:S421-426.
Sabroe I, Jones EC, Whyte MK, Dower SK. 2005b. Regulation of human neutrophil chemokine receptor expression and function by activation of Toll-like receptors 2 and 4. Immunology 115(1):90-98.
Sabroe I, Prince LR, Jones EC, Horsburgh MJ, Foster SJ, Vogel SN, Dower SK, Whyte MK. 2003. Selective roles for Toll-like receptor (TLR)2 and TLR4 in the regulation of neutrophil activation and life span. J Immunol 170(10):5268-5275.
Saegusa S, Totsuka M, Kaminogawa S, Hosoi T. 2009. Saccharomyces cerevisiae and Candida albicans stimulate cytokine secretion from human neutrophil-like HL-60 cells differentiated with retinoic acid or dimethylsulfoxide. Biosci Biotechnol Biochem 73(12):2600-2608.
Schipper RG, Penning LC, Verhofstad AA. 2000. Involvement of polyamines in apoptosis. Facts and controversies: effectors or protectors? Semin Cancer Biol 10(1):55-68.
Shuto T, Furuta T, Cheung J, Gruenert DC, Ohira Y, Shimasaki S, Suico MA, Sato K, Kai H. 2007. Increased responsiveness to TLR2 and TLR4 ligands during dimethylsulfoxide-induced neutrophil-like differentiation of HL-60 myeloid leukemia cells. Leuk Res 31(12):1721-1728.
Shyu KG, Wang BW, Lin CM, Chang H. Cyclic stretch enhances the expression of toll-like receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-kappaB pathway. J Biomed Sci 17:15.
Swanson BN. 1985. Medical use of dimethyl sulfoxide (DMSO). Rev Clin Basic Pharm 5(1-2):1-33.
Tabor CW, Tabor H. 1984. Polyamines. Annu Rev Biochem 53:749-790.
Tabor CW, Tabor H. 1985. Polyamines in microorganisms. Microbiol Rev 49(1):81-99.
Takashima K, Matsunaga N, Yoshimatsu M, Hazeki K, Kaisho T, Uekata M, Hazeki O, Akira S, Iizawa Y, Ii M. 2009. Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model. Br J Pharmacol 157(7):1250-1262.
Tamassia N, Le Moigne V, Calzetti F, Donini M, Gasperini S, Ear T, Cloutier A, Martinez FO, Fabbri M, Locati M, Mantovani A, McDonald PP, Cassatella MA. 2007. The MyD88-independent pathway is not mobilized in human neutrophils stimulated via TLR4. J Immunol 178(11):7344-7356.
Thao D. 2009. 最新圖解免疫學. 林春福, translator. 台北市: 合記圖書出版社. 25-170 p.
Thomas T, Thomas TJ. 2001. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci 58(2):244-258.
Tsatsanis C, Androulidaki A, Alissafi T, Charalampopoulos I, Dermitzaki E, Roger T, Gravanis A, Margioris AN. 2006. Corticotropin-releasing factor and the urocortins induce the expression of TLR4 in macrophages via activation of the transcription factors PU.1 and AP-1. J Immunol 176(3):1869-1877.
Vallabhapurapu S, Karin M. 2009. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol 27:693-733.
Wallace HM, Fraser AV, Hughes A. 2003. A perspective of polyamine metabolism. Biochem J 376(Pt 1):1-14.
West AP, Koblansky AA, Ghosh S. 2006. Recognition and signaling by toll-like receptors. Annu Rev Cell Dev Biol 22:409-437.
Yamashiro S, Kamohara H, Wang JM, Yang D, Gong WH, Yoshimura T. 2001. Phenotypic and functional change of cytokine-activated neutrophils: inflammatory neutrophils are heterogeneous and enhance adaptive immune responses. J Leukoc Biol 69(5):698-704.
Zhang M, Caragine T, Wang H, Cohen PS, Botchkina G, Soda K, Bianchi M, Ulrich P, Cerami A, Sherry B, Tracey KJ. 1997. Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response. J Exp Med 185(10):1759-1768.
Zhang W, Wang LZ, Yu JT, Chi ZF, Tan L. 2011. Increased expressions of TLR2 and TLR4 on peripheral blood mononuclear cells from patients with Alzheimer''s disease. J Neurol Sci 315(1-2):67-71.
王恩華主編. 2005. 病理學. 台北縣中和市: 新文京開發. 92-118 p.
何敏夫編著. 1995. 血液學. 台北市: 合記圖書出版社. 7-28 p.
林嘉祥. 2010. 鳥胺酸去羧化酶降低二甲亞碸所誘導類嗜中性白血球分化、活化及細胞凋亡， 碩士論文. 台中市: 中山醫學大學醫學研究所. 12-38 p.
曾哲明編著. 2005. 免疫學. 台北縣中和市: 新文京開發. 320-327 p.