跳到主要內容

臺灣博碩士論文加值系統

(44.192.22.242) 您好!臺灣時間:2021/08/01 13:45
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:林伯峰
研究生(外文):Bo-FengLin
論文名稱:點帶石斑魚兩腫瘤壞死因子-α (TNF-α1、TNF-α2) 蛋白功能性分析之探討
論文名稱(外文):Functional analysis of TNF-α1 and TNF-α2 of orange-spotted grouper (Epinephelus coioides)
指導教授:林翰佑
指導教授(外文):Han-You Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
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:103
中文關鍵詞:點帶石斑魚腫瘤壞死因子-α細胞激素
外文關鍵詞:Epinephelus coioidestumor necrosis factor- alpha (TNF-α)cytokine
相關次數:
  • 被引用被引用:5
  • 點閱點閱:292
  • 評分評分:
  • 下載下載:17
  • 收藏至我的研究室書目清單書目收藏:0
先天免疫反應是魚類抵抗疾病的第一道防線,許多細胞激素(cytokine) 參與其中,像是腫瘤壞死因子-α (TNF-α) 就是一個非常多效性的細胞激素,也是TNF superfamily中,相當具有代表性的蛋白質之ㄧ,它由數個相似結構和功能的蛋白質所組成,並且由數種免疫細胞製造,因此它在免疫系統及前發炎反應中都扮演著相當重要的角色,因為它不但能夠刺激免疫細胞並活化一連串的免疫反應,也能調控細胞走向細胞凋亡 (apoptosis) 的命運。除此之外,也可將TNF-α當作魚隻被病原菌感染刺激之後,初期免疫反應發生時的指標細胞激素。在我們過去的研究中,已經成功選殖到點帶石斑魚兩個TNF-α基因,mRNA長度分別為1516 bp與1530 bp,都具有4 exons及3個introns,胺基酸的相似度僅有38%。而這兩個TNF-α基因在石斑魚受到LPS刺激下,各免疫臟器有著不同的表現量,所以推測此兩個基因應該具備不同的調控路徑和生物功能。本研究中,將進一步探討這兩個TNF-α蛋白其生物功能性的異同,實驗以大腸桿菌表現的兩個rgTNF-α1、rgTNF-α2蛋白作為實驗材料。由原態膠體電泳分析發現rgTNF-α1與哺乳類TNF-α相同,皆具有單聚體、二聚體、三聚體多種形式。兩rgTNF-α1、rgTNF-α2蛋白利用來刺激點帶石斑魚兩種不同的周邊血液白血球,發現角狀周邊血液白血球在rgTNF-α1和rgTNF-α2蛋白刺激之下,皆能誘導tnf-α1、tnf-α2、il-6、il-1β、ccl4、mx基因表現,但caspase 9只會被rgTNF-α2誘導,但在巨噬細胞中,除了皆能誘導tnf-α1、tnf-α2、il-6、il-1β、ccl4基因表現之外,rgTNF-α1尚能誘導caspase 9的表現。結果顯示兩個rgTNF-α對於不同的周邊血液白血球,可能有不同的調控功能。同時將兩rgTNF-α分別免疫兔子、老鼠取得抗石斑魚TNF-α1及TNF-α2之特異性多株抗體,更進一步作為TNF-α特異性
抑制劑,以釐清gTNF-α1和gTNF-α2蛋白功能。

The innate immune response is the first line of fish defense against pathogens. Many cytokines involved in innate immunity including tumor necrosis factor-α (TNF-α), which is a very pleiotropic cytokine. The TNF-α is the most representative TNF in its superfamily and consisted of many proteins with similar structures and functions. The TNF-α is manufactured by several kinds of immune cells and plays an important role in the immune system and pro-inflammatory response. It stimulates not only immune cells and activates a series of immune responses, but also leads to apoptosis. In addition, TNF-α expressed can be considered as the indicators of immune response in the early stage of infection. In our previous studies, we had successfully cloned two grouper TNF-α gene and presented the gene structure. The open reading frames of both genes are approximately 1.5 kb in length and organized into four exons and three introns, being only 38% identical at the amino acid level. Both gTNF-α genes have different expression pattern in fish immune organs under LPS stimulation. Therefore, we assume that these two genes should be involved in different pathways and have different biological functions.
The non-reducing SDS-PAGE results showed that rgTNF-α1 expression in E. coli contains monomer, dimer and trimer forms as the nature formed mammal TNF-α. Both rgTNF-αs were used to stimulate the grouper angular peripheral blood leukocytes (PBLs) and PBL-derived macrophahe-like cell. Results revealed that both the rgTNF-αs induced tnf-α1, tnf-α2, il-6, il-1β, ccl4 and mx gene expression, but caspase 9 only be induced by rgTNF-α2 in angular PBLs. The stimulation of macrophage-like cell with both rgTNF-αs resulted in increased tnf-α1, tnf-α2, il-6, il-1β, ccl4 expression, besides, rgTNF-α1 also induced caspase 9 expression. Together, these results indicate that rgTNF-α1 and rgTNF-α2 have different regulatory function in different PBLs.
Moreover, rabbits and mice were immunized with rgTNF-α1 or rgTNF-α2 to obtain specific polyclonal antibodies. Therefore, we can use of TNF-α antibody inhibiting the rgTNF-αs function in PBLs and analysis of these immune-related genes which is mainly regulated by the gTNF-α1 or gTNF-α2 protein.

目錄
中文摘要...................................................Ⅰ
Abstract................................................ III
目錄..................................................... IV
圖目錄..................................................... V
壹、研究背景.................................................1
1-1點帶石斑魚............................................1
1-2魚類免疫系統...........................................2
1-3腫瘤壞死因子-α.........................................7
貳、研究目的................................................16
參、實驗材料與方法...........................................17
肆、實驗結果................................................35
伍、討論...................................................41
陸、參考文獻................................................52
柒、圖表...................................................60


Aggarwal, B. B., W. J. Kohr, et al. (1985). Human tumor necrosis factor. Production, purification, and characterization. J Biol Chem 260(4): 2345-2354.

Aggarwal, B. B. (2003). Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 3(9): 745-756.

Allenbach, C., P. Launois, et al. (2008). An essential role for transmembrane TNF in the resolution of the inflammatory lesion induced by Leishmania major infection. Eur J Immunol 38(3): 720-731.

Batten, P., M. H. Yacoub, et al. (1996). Effect of human cytokines (IFN-gamma, TNF-alpha, IL-1 beta, IL-4) on porcine endothelial cells: induction of MHC and adhesion molecules and functional significance of these changes. Immunology 87(1): 127-133.

Baud, V. and M. Karin (2001). Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol 11(9): 372-377.

Beltinger, C. P., P. S. White, et al. (1996). Physical mapping and genomic structure of the human TNFR2 gene. Genomics 35(1): 94-100.

Boshra, H., J. Li, et al. (2006). Recent advances on the complement system of teleost fish. Fish Shellfish Immunol 20(2): 239-262.

Bridle, A. R., R. N. Morrison, et al. (2006). The expression of immune-regulatory genes in rainbow trout, Oncorhynchus mykiss, during amoebic gill disease (AGD). Fish Shellfish Immunol 20(3): 346-364.

Browning, J. L., I. Dougas, et al.(1995). Characterization of surface lymphotoxin forms. Use of specific monoclonal antibodies and soluble receptors. J Immunol 154(1): 33-46.

Buchmann, K. (2001). Lectins in fish skin: do they play a role in host-monogenean interactions? J Helminthol 75(3): 227-231.

Byeon, H. E., S. H. Um, et al. (2012). Ohioensin F suppresses TNF-alpha-induced adhesion molecule expression by inactivation of the MAPK, Akt and NF-kappaB pathways in vascular smooth muscle cells. Life Sci 90(11-12): 396-406.

Chang, L. and M. Karin (2001). Mammalian MAP kinase signalling cascades. Nature 410(6824): 37-40.

Chang, S. F., G. H. Ngoh-Lim, et al. (2002). Initial investigations into two viruses isolated from marine food fish in Singapore. Vet Rec 150(1): 15-16.

Chaplin, D. D. (2010). Overview of the immune response. J Allergy Clin Immunol 125(2 Suppl 2): S3-23.

Corti, A., G. Fassina, et al. (1992). Oligomeric Tumor-Necrosis-Factor-Alpha Slowly Converts into Inactive Forms at Bioactive Levels. Biochem J 284: 905-910.

Duncan, R. J. S., P. D. Weston, et al. (1983). A New Reagent Which May Be Used to Introduce Sulfhydryl-Groups into Proteins, and Its Use in the Preparation of Conjugates for Immunoassay. Anal Biochem 132(1): 68-73.

Englaro, W., P. Bahadoran, et al. (1999). Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation. Oncogene 18(8): 1553-1559.

Esparza, I., D. Mannel, et al. (1987). Interferon gamma and lymphotoxin or tumor necrosis factor act synergistically to induce macrophage killing of tumor cells and schistosomula of Schistosoma mansoni. J Exp Med 166(2): 589-594.

Fishman, M. (1991). Cytolytic activities of activated macrophages versus paraformaldehyde-fixed macrophages; soluble versus membrane-associated TNF. Cell Immunol 137(1): 164-174.

Garcia-Castillo, J., P. Pelegrin, et al. (2002). Molecular cloning and expression analysis of tumor necrosis factor alpha from a marine fish reveal its constitutive expression and ubiquitous nature. Immunogenetics 54(3): 200-207.

Garcia-Garcia, E., R. Rosales, et al. (2002).
Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase are recruited for Fc receptor-mediated phagocytosis during monocyte-to-macrophage differentiation.
J Leukoc Biol 72(1): 107-114.

Goetz, F. W., J. V. Planas, et al. (2004). Tumor necrosis factors. Dev Comp Immunol 28(5): 487-497.

Gomez, G. D. and J. L. Balcazar (2008). A review on the interactions between gut microbiota and innate immunity of fish. FEMS Immunol Med Microbiol 52(2): 145-154.

Grayfer, L., J. G. Walsh, et al. (2008). Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha. Dev Comp Immunol 32(5): 532-543.

Grayfer, L. and M. Belosevic (2009). Molecular characterization of tumor necrosis factor receptors 1 and 2 of the goldfish (Carassius aurutus L.). Mol Immunol 46(11-12): 2190-2199.

Grell, M., E. Douni, et al. (1995). The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 83(5): 793-802.

Gruber, M. F. and T. L. Gerrard (1992). Production of macrophage colony-stimulating factor (M-CSF) by human monocytes is differentially regulated by GM-CSF, TNF alpha, and IFN-gamma. Cell Immunol 142(2): 361-369.

Hansen, T. E. and J. B. Jorgensen (2007). Cloning and characterisation of p38 MAP kinase from Atlantic salmon A kinase important for regulating salmon TNF-2 and IL-1beta expression. Mol Immunol 44(12): 3137-3146.

Havell, E. A. (1989). Evidence that tumor necrosis factor has an important role in antibacterial resistance. J Immunol 143(9): 2894-2899.

Hiasa, M., M. Abe, et al. (2009). GM-CSF and IL-4 induce dendritic cell differentiation and disrupt osteoclastogenesis through M-CSF receptor shedding by up-regulation of TNF-alpha converting enzyme (TACE). Blood 114(20): 4517-4526.

Hirono, I., B. H. Nam, et al. (2000). Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus. J Immunol 165(8): 4423-4427.

Hober, D., A. Haque, et al. (1989). Production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) in patients with AIDS. Enhanced level of TNF-alpha is related to a higher cytotoxic activity. Clin Exp Immunol 78(3): 329-333.

Kadowaki, T., H. Harada, et al. (2009). Two types of tumor necrosis factor-alpha in bluefin tuna (Thunnus orientalis) genes: Molecular cloning and expression profile in response to several immunological stimulants. Fish Shellfish Immunol 27(5):

Kawasaki, H., R. Onuki, et al. (2002). Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries. Nat Biotechnol 20(4): 376-380.

Kollias, G. and D. Kontoyiannis (2002). Role of TNF/TNFR in autoimmunity: specific TNF receptor blockade may be advantageous to anti-TNF treatments. Cytokine Growth Factor Rev 13(4-5): 315-321.

Lam, F. W., S. Y. Wu, et al. (2011). The expression of two novel orange-spotted grouper (Epinephelus coioides) TNF genes in peripheral blood leukocytes, various organs, and fish larvae. Fish Shellfish Immunol 30(2): 618-629.

Lam, K. S., P. Scuderi, et al. (1988). Analysis of the molecular organization of recombinant human tumor necrosis factor (rTNF) in solution using ethylene glycolbis(succinimidylsuccinate) as the cross-linking reagent. J Biol Response Mod 7(3): 267-275.

Lieschke, G. J. and N. S. Trede (2009). Fish immunology. Curr Biol 19(16): R678-682.

Li, J., D. R. Barreda, et al. (2006). B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities. Nat Immunol 7(10): 1116-1124.

Locksley, R. M., N. Killeen, et al. (2001). The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104(4): 487-501.

Luksa, J., V. Menart, et al. (1994). Purification of Human Tumor-Necrosis-Factor by Membrane Chromatography. Journal of Chroma A 661(1-2): 161-168.

MacKenzie, S., J. V. Planas, et al. (2003). LPS-stimulated expression of a tumor necrosis factor-alpha mRNA in primary trout monocytes and in vitro differentiated macrophages. Dev Comp Immunol 27(5): 393-400.

Magnadottir, B. (2006). Innate immunity of fish (overview).
Fish Shellfish Immunol 20(2): 137-151.

Narhi, L. O. and T. Arakawa (1987). Dissociation of Recombinant Tumor-Necrosis-Factor-Alpha Studied by Gel-Permeation Chromatography. Biochem Biophys Res Commun 147(2): 740-746.

Nilsson, G., M. Carlsson, et al. (1994). TNF-alpha and IL-6 induce differentiation in the human basophilic leukaemia cell line KU812. Immunology 81(1): 73-78.

Nomiyama, H., K. Hieshima, et al. (2008). Extensive expansion and diversification of the chemokine gene family in zebrafish: identification of a novel chemokine subfamily CX. BMC Genomics 9: 222.

O'Malley, W. E., B. Achinstein, et al. (1963). Action of Bacterial Polysaccharide on Tumors. Iii. Repeated Response of Sarcoma 37, in Tolerant Mice, to Serratia Marcescens Endotoxin. Cancer Res 23: 890-895.

Ordas, M. C., M. M. Costa, et al. (2007). Turbot TNFalpha gene: molecular characterization and biological activity of the recombinant protein. Mol Immunol 44(4): 389-400.

Park, C. I., T. Kurobe, et al. (2003). Cloning and characterization of cDNAs for two distinct tumor necrosis factor receptor superfamily genes from Japanese flounder Paralichthys olivaceus. Dev Comp Immunol 27(5): 365-375.

Petersen, C. M., A. Nykjaer, et al. (1989). Bioactive human recombinant tumor necrosis factor-alpha: an unstable dimer? Eur J Immunol 19(10):1887-1894

Pfeffer, K., T. Matsuyama, et al. (1993). Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 73(3): 457-467.

Pinto, L. M., S. A. Oliveira, et al. (1999). Increased pro-inflammatory cytokines (TNF-alpha and IL-6) and anti-inflammatory compounds (sTNFRp55 and sTNFRp75) in Brazilian patients during exanthematic dengue fever. Mem Inst Oswaldo Cruz 94(3): 387-394.

Postma, N. S., R. C. Hermsen, et al. (1999). Thiolated recombinant human tumor necrosis factor-alpha protects against Plasmodium berghei K173-induced experimental cerebral malaria in mice. Antimicrob Agents Chemother 43(5): 1027-1033.

Richter, C., S. Messerschmidt, et al. (2012). The Tumor Necrosis Factor Receptor Stalk Regions Define Responsiveness to Soluble versus Membrane-Bound Ligand. Mol Cell Biol 32(13): 2515-2529.

Robertsen, B. (2006). The interferon system of teleost fish. Fish Shellfish Immunol 20(2): 172-191.

Roca, F. J., I. Mulero, et al. (2008). Evolution of the inflammatory response in vertebrates: fish TNF-alpha is a powerful activator of endothelial cells but hardly activates phagocytes. J Immunol 181(7): 5071-5081.

Rombout, J. H., H. B. Huttenhuis, et al. (2005). Phylogeny and ontogeny of fish leucocytes. Fish Shellfish Immunol 19(5): 441-455.


Rothe, J., W. Lesslauer, et al. (1993). Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364(6440): 798-802.

Saeij, J. P., R. J. Stet, et al. (2003). Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance? Dev Comp Immunol 27(1): 29-41.

Saurabh, S. and P. K. Sahoo (2008). Lysozyme: an important defence molecule of fish innate immune system. Aquacult Res 39(3): 223-239.

Savan, R. and M. Sakai (2004). Presence of multiple isoforms of TNF alpha in carp (Cyprinus carpio L.): genomic and expression analysis. Fish Shellfish Immunol 17(1): 87-94.

Savan, R., T. Kono, et al. (2005). A novel tumor necrosis factor (TNF) gene present in tandem with theTNF-alpha gene on the same chromosome in teleosts. Immunogenetics 57(1-2): 140-150.

Simon, A., J. Fah, et al. (1991). Interferon-regulated Mx genes are not responsive to interleukin-1, tumor necrosis factor, and other cytokines. J Virol 65(2): 968-971.

Smith, R. A. and C. Baglioni (1987). The Active Form of Tumor-Necrosis-Factor Is a Trimer. Journal of Biol Chem
262(15): 6951-6954.

Tsutsui, S., M. Yamaguchi, et al. (2009). Common skate (Raja kenojei) secretes pentraxin into the cutaneous secretion: The first skin mucus lectin in cartilaginous fish. J Biochem 146(2): 295-306.

Van Hauwermeiren, F., R. E. Vandenbroucke, et al. (2011).
Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1. Cytokine Growth Factor Rev 22(5-6): 311-319.

Watts, M., B. L. Munday, et al. (2001). Immune responses of teleost fish. Aust Vet J 79(8): 570-574.

Weinberg, J. B., M. A. Misukonis, et al. (1995). Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages. Blood 86(3): 1184-1195.

Zou, J., C. J. Secombes, et al. (2003). Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus). Dev Comp Immunol 27(10): 845-858.


連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top