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系統識別號 U0026-0812200910433739
論文名稱(中文) Jurkat細胞的介白素10號基因啟動子上之 轉錄因子結合位調節介白素10號表現之研究
論文名稱(英文) The Transcription Element of IL-10 promoter Mediating the Expression of IL-10 in Jurkat Cells
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 91
學期 2
出版年 92
研究生(中文) 林亨鍇
研究生(英文) Heng-Kai Lin
學號 s4688111
學位類別 碩士
語文別 中文
論文頁數 54頁
口試委員 指導教授-楊倍昌
召集委員-許秉寧
口試委員-林以行
口試委員-陳文杰
中文關鍵字 腦瘤細胞株  聚合連鎖反應 
英文關鍵字 IL-10  promoter  electrophoresis mobility shift assay  forskolin  Jurkat  Fas-FasL  signal transduction pathway 
學科別分類
中文摘要 IL-10在免疫調節上佔有非常重要而關鍵的角色, Jurkat細胞不論以forskolin刺激或將之與腦瘤細胞株U-118MG共同培養後都會導致IL-10表現量增加。我們想進一步了解Jurkat細胞的IL-10基因在轉錄上的調控。首先,我們在IL-10 promoter中挑選了二段基因片段,以聚合酶連鎖反應﹝PCR﹞放大,作為探針﹝probe﹞。利用electrophoresis mobility shift assay (EMSA) 試驗,分析有哪些轉錄因子與IL-10表現有關。結果發現Jurkat與BJAB二種細胞之核萃取物與IL-10 promoter片段間的結合能力並不相同。不論是以forskolin刺激或與腦瘤細胞株U-118MG共同培養後的Jurkat細胞的核萃取液,與IL-10 promoter上-1120 ~ -986段核酸雜交作EMSA試驗,結果顯示有一個未受刺激前存在的特異性的蛋白質-核酸複合物(稱之為E band)在此二種處理後消失了,顯示出此段基因可能含有一個負調控因子。forskolin的刺激會導致CRE的結合活性、CREB磷酸化以及IL-10的mRNA增加,而這些forskolin 的作用則受到PKA的抑制劑-KT5720抑制。在與U-118MG共同培養下,CREB磷酸化的表現並不明顯,顯示出forskolin經由PKA這條路徑引致Jurkat細胞表現IL-10,而U-118MG則否。同時也發現U-118MG誘使Jurkat細胞IL-10表現量增加與Fas-FasL所傳入的訊號有關,但是此signal transduction pathway是否包含caspase 8以及caspase 3活化,仍有待進一步研究。
英文摘要 IL-10 plays a crucial role in immune regulation. Our previous study showed that IL-10 level was increased in Jurkat cells by coculture with U-118MG or forskolin-treatment. In this study, we intended to investigate the regulation of IL-10 gene in Jurkat cells in detail. Two fragments of IL-10 promoter were amplified with polymerase chain reaction (PCR) and used as probes in electrophoresis mobility shift assay (EMSA) to analyze the involved transcription factors for the transcription of IL-10 gene. EMSA data showed that nuclear extracts of Jurkat and BJAB cells had different binding capabilities on IL-10 promoter region. A particular band, designated as E, being a complex of nuclear extracts and IL-10 promoter region from –1120 to –986, disappeared after treated with forskolin or coculture with U-118MG. It implied a negative regulatory role of this complex on IL-10 gene. PKA inhibitor (KT5720) can inhibit the CREB phosphorylation and IL-10 production. In addition, treatment with forskolin enhanced CREB phosphorylation (Western immunoblot) and CRE binding activity (EMSA) in Jurkat cell. However, coculture with U-118MG did not significantly induce the phosphorylation of CREB and CRE
binding activity in Jurkat cells. Therefore, PKA may not be the major pathway for IL-10 expression in Jurkat cells upon coculture with U-118MG. Moreover, we found that casepase 8 might not involve in U-118MG induced IL-10 production. In summary, our results indicated that direct activation of PKA pathway can stimulate the transcription of IL-10 gene. However, PKA pathway does not mediate U-118MG induced IL-10 production.
論文目次 總目錄
總目錄………………………………………………………………………i
中文摘要………………………………………………………………… ii
英文摘要……………………………………………………………………iv
圖目錄………………………………………………………………………vi
縮寫對照表……………………………………………………………… vii
誌謝………………………………………………………………………viii
緒論…………………………………………………………………………01
實驗設計……………………………………………………………………05
材料與方法…………………………………………………………………07
結果…………………………………………………………………………24
討論…………………………………………………………………………29
參考文獻……………………………………………………………………34
圖……………………………………………………………………………41
自述…………………………………………………………………………54
參考文獻 何維修。The Fas-L on glioma cells affect the expression of cytokines and viability in immune cells.國立成功大學微生物及免疫學研究所碩士論文。中華民國,台南,民91。
黃俊淵。利用增強型綠色螢光蛋白報告基因研究介白素十號的啟動子調控。國立成功大學微生物及免疫學研究所碩士論文。中華民國,台南,民88。
Aman, M.J., Tretter, T., Eisenbeis, I., Bug, G., Decker, T., Aulitzky, W.E., Tilg, H., Huber, C. and Peschel, C. Interferone-alpha stimulates production of interleukine-10 in activated CD4+ T cells and monocytes. Blood 87:4731-4736,1996.
Andrews N. C. and Faller D. V. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 19:2499, 1991.
Aruga A., Aruga E., Tanigawa K., Bishop D.K., Sondak V.K. and Chang A.E. Type 1 versus type 2 cytokine release by VβT cell subppulations determines in vivo antitumor reactivity:IL-10 mediates a suppressive role. J. Immunol. 159:664-673,1997.
Badou A., Bennasser Y., Moreau M., Leclerc C., Benkirane M., and Bahraoui E. Tat protein of human immunodeficiency virus type I induces interleukone-10 in human peripheral blood monocytes:implication of protein kinase C-dependent pathway. J.Virol. 74:10551-10562, 2000.
Benjamin D., Knobloch T.J. and Dayton M.A. Human B-cell interleukine–10:B-cell lines derived from patients with acquired immunodeficiency syndrome and Burkitt’s lymphoma constitutively secrete large quantities of interleukine-10. Blood 80:1289-12298,1992.
Book A. A., Fielding K. E., Kundu N., Wilson M. A., Fulton A. M. and Laterra J. IL-10 gene transfer to intracranial 9L glioma:tumor inhibition and cooperation with IL-2. J. Neuroimmunol. 92:50-59,1998
Brenner S., Prösch S., Schenke—Layland K., Riese U., Gausmann U., and Platzer C. cAMP-induced interleukine-10 promoter activation dependents on CCAAT/enhancer-binding protein expression and monocytic differentiation. J. Biol.Chem. 278:5597-5604, 2003.
Brightbill H.D. Plevy S.E. Modlin R.L. Smale S.T. A prominent role for SP-1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophage. J.Immunol. 164:1940-1951, 2000.
Daigle I., Rückert B., Schnetzler G. and Simon H.U. Induction of the IL-10 gene via the Fas receptor in monocytes-an anti-inflammatory mechanism in the absence of apoptosis. Eur. J.Immunol. 30:2991-2997,2000.
Donnelly R.P., Freeman S.L., and Hayes M.P. Inhibition of IL-10 expression by IFN-γ up regulated transcription of TNF-α in human monocyte. J. Immunol. 155:1420-1427, 1995.
Eskdale J., Kube D., Tesch H., Gallagher G. Mapping of the human IL-10 gene and further characterization of the 5’flanking sequence. Immunogenetics 46:120-128,1997.
Foey A. D., Parry S. L., Williann L. M., Feldmann M., Foxwell B.M.J.and Brennan F.M. Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-α:Role of the p38 and p42/44 mitogen-activated protein kinase. J.Immunol. 160:920-928, 1998.
Gary-Gouy H., Harriague J., Bismuth G., Platzer C., Schmitt C., and Dalloul A. H. Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production. Blood,100:4537-4543,2002.
Gerosa, F., Paganin, C., Peritt, D., Paiola, F., Scupoli, M.t. Asteamezaga, M., Frank, I. and Trinchieri, G. Interleukine 12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interkeukine-10. J. Exp. Med.183:2559-2569, 1996.
Halak B.K., Maguire H. C., Jr., and Lattime E. C. Tumor-induced interleukine-10 inhibits type 1 immune responses directed at a tumor antigen as well as a non tumor antigen present at the tumor site. Cancer Research 59:911-917,1999.
Hou, J., Baichwal, V. and Cao, Z. Regulatory elements and transcription factors controlling basal and cytokine-induced expression of the gene encoding intracellular adhesion molecule. Proc Natl Acad Sci USA 91:11641-11645,1994.
Huettner C., Czub S., Kerkau S., Roggendorf W. and Tonn J.C. Interleukin 10 is expressed in human gliomas in vivo and increases glioma cell proliferation and motility in vitro. Anticancer Research 17:3217-3244,1997.
Kim J. M., Brannan C. I., Copeland N. G., Jenkins N. A., Khan T. A. and Moore K. W. Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes. J. Immunol. 148:3618-3623,1992.
Kim J., Modlin R. L., Moy R. L., Dubinett S. M., McHugh T., Nickoloff B. J., and Uyemura K. IL-10 production in cutaneous basal and squamous cell carcinomas. J. Immunol. 155:2240-2247,1995.
Kube D., Platzer C., von Knethen A., Straub H., Bohlen H., Hafner M., Tesch H. Isolation of the human interleukine 10 promoter. Characterization of the promoter activity in Burkitt’s lymphoma cell line. Cytokine 7:1-7,1995.
Mantovani R. The molecular biology of the CCAAT-binding factor NF-Y. Gene 239: 15-17,1999.
Meisel C., Vogt K., Platzer C., Randow F., Liebenthal C., and Volk H.D.. Differential regulation of monocytic tumor necrosis factor-α and interleukine 10 expression. Eur. J. Immunol. 26:1580-1586 ,1996.
Meyer, C.F., Wang, X., Chang, C., Templeton, D., and Tan, T.H. Interaction between c-Rel and the mitogen-activated protein kinase I signaling cascade in mediating kappaB enhancer activation. J. Biol. Chem. 271:8971-8976, 1996.
Nakajima T., Uchida C., Anderson S. F., Parvin J. D., and Montminy M. Analysis of a cAMP-responsive activator reveals a two-component mechanism for transcriptional induction via signal-dependent.Genes & Dev. 11:738-747, 1997.
Ohmori, Y. and Hamilton, T.A. The interfero-stimulated response element and a kappa-B site mediate synergistic induction of the murine IP-10 gene transcription by IFN-gamma and TNF-alpha. J. Immunol. 154:5235- 5244,1995.
Platzer C., Fritsch E., Elsner T., Lehmann M. H., Volk H.D. and Prösch S. Cyclic adenosine monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells. Eur. J.Immunol. 29:3098-3104,1999.
Reiling N., Blumenthal A., Flad H.D., Ernst M. and Ehlers S. Mycobacteria-induced TNF-alpha and IL-10 formation by human macrophage is differentially regulated at the level of mitogen-activated protein kinase activity. J.Immunol. 167:3339-3345, 2001.
Shaw T. C., Thomas L. H., Friedland J.S. Regulation of IL-10 secretion after phagocytosis of Mycobacterium tuberculosis by human monocytic cells. Cytokine 12:483-486,2000.
Steinbrink K., Jonuleit H., Müller G., Schuler G., Knop J., and Enk A. H. Interleukin-10-treated human dendritic cells induce a melanoma-antigen specific anergy in CD8+ T cells resulting in a failure to lyse tumor cells. Blood 93:1634-642,1999.
Suárez A., Castro P., Alonso R., Mozo L., and Gutiérrez C. Interindividual variations in constitutive interleukine-10 messenger RNA and protein levels and their association with genetic polymorphisms. Transplantation 75:711-717, 2003.
Tone M., Powell M. J., Tone Y., Thompson S.A.J., and Waldmann H. IL-10 gene expression is controlled by the transcription factors SP-1 and SP-3. J.Immunol. 165:286-291, 2000.
Wagner S., Czub S., Greif M., Vince G. H., Süss N., Kerkau S., Rieckmann P., Roggendorf W., Roosen K. and Tonn J.C. Microglial/Macrophage expression of interleukine 10 in human glioblastomas. Int. J. Cancer 82:12-16, 1999.
Walker W. H., Fucci L., and Habener J. F. Expression of the gene encoding transcription factor cyclic adenosine 3’,5’-monophosphate(cAMP)response element-binding protein(CREB):Regulation by follicle primary rat sertoli cells. Endocrinology 136:3534-3545, 1995.
Wang C.Y., Mayo M. W., Korneluk R. G., Goeddel D.V., Baldwin A. S. Jr. NF-κB antiappoptosis:Induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281:1680-1683, 1998.
Wanidworanum C. and Strober W. Predominant role of tumor necrosis factor-α in human monocyte IL-10 synthesis. J. Immunol. 151:6853-6861,1993.
Windhagen, A., Anderson, D.E., Caririzosa, A., Williams, R.E. and Hafer, D.A. IL-12 induces human T-cells secreting IL-10 with IFN-gamma. J. Immunol. 157:1127-1131,1996.
Witsch E. J., Peiser M., Hutloff A., Büchner K., Dorner B. G., Jonulit H., Mages H. W. and Kroczek R. A.. ICOS and CD28 reversely regulate IL-10 on re-activation of human effector T cells with mature dendritic cells. Eur. J. Immunol. 32:2680-2686,2002.
Wölfl S., Martinez C., and Majzoub J.A. Inducible binding of cyclic adenosine 3’,5’-monophosphate(cAMP)-responsive element binding protein(CREB)to a cAMP-responsive promoter in vivo. Mol. Endo. 13:659-668,1999.
Yamamoto K.K., Gonzalez G.A., Biggs III W.H. and Montminy M.R. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334:494-498,1988.
Zou J.P., Morford L.A., Chougnet C., Dix A.R., Brooks A.G., Torres N., Shuman J.D., Coligan J.E., Brooks W.H., Roszman T.L. and ShearerG.M.Human glioma-inducedimmunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers. J.Immunol.162:4882-4892,1999.
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