跳到主要內容

臺灣博碩士論文加值系統

(44.192.95.161) 您好!臺灣時間:2024/10/16 02:48
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

我願授權國圖
: 
twitterline
研究生:林玉苑
研究生(外文):Yu-Yuan Lin
論文名稱:巨噬細胞中抑制PDE4對LPS誘導發炎反應之調控
論文名稱(外文):Regulation of LPS-induced inflammatory responses by PDE4 inhibitor in macrophages
指導教授:金秀蓮
指導教授(外文):S.-L. Catherine Jin
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:57
中文關鍵詞:反應性含氧物種腫瘤壞死因子Src 酪胺酸酶磷酸雙酯酶脂多糖
外文關鍵詞:LPSPDE4Src tyrosine kinaseTNF-aROS
相關次數:
  • 被引用被引用:0
  • 點閱點閱:476
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
第四型環狀核甘酸磷酸二酯酶 (PDE4) 為一可以水解第二傳訊者cAMP的酵素,因此有調節細胞中cAMP濃度與訊息傳遞的功能。在免疫細胞中,PDE的表現以PDE4為主,且實驗證實抑制PDE4活性有降低發炎反應的作用,故此類酵素被認為是免疫細胞功能的重要調節者。PDE4酵素家族包含四個基因,PDE4A、PDE4B、PDE4C與PDE4D。先前研究指出LPS刺激巨噬細胞可增加細胞內PDE4B的表現與活性,並釋放大量的TNF-?,而PDE4抑制劑rolipram會顯著抑制TNF-?的產生。在LPS活化TLR4同時也會增加細胞中Src tyrosine kinase的活性,給予Src tyrosine kinase抑制劑PP2亦可降低LPS誘導TNF-?的釋放。本研究結果顯示,在Raw 264.7細胞中,處理PP2會使LPS誘導TNF-?的釋放降低約50%,而rolipram與db-cAMP則幾乎可完全抑制此TNF-?的反應;在PDE4B基因剔除鼠的巨噬細胞中,LPS刺激TNF-?的釋放量會有明顯的下降,其降低程度與rolipram或PP2抑制野生型小鼠巨噬細胞的程度相當,顯示PDE4B可能藉由調節cAMP與Src tyrosine kinase的訊息傳導路徑以調控LPS/TNF-?的反應。我們發現在Raw 264.7細胞中,PP2不會抑制LPS所誘導的PDE4活性,這結果排除了PP2抑制TNF-?的釋放可能是由於其抑制PDE4B活性的可能性。LPS刺激巨噬細胞會使細胞內ROS增加,而此反應可被cAMP與PKA所抑制,但是抑制Src tyrosine kinase活性却不會影響ROS的產生。在Raw 264.7細胞中,LPS對Lyn-tyr396的磷酸化沒有明顯影響,而Lyn-tyr507的磷酸化卻有抑制的現象,同時,rolipram對Lyn的磷酸化表現也與預期不符。綜合本研究結果我們發現,在巨噬細胞中,抑制PDE4B可以降低LPS刺激的TNF-?與ROS產生,而抑制Src tyrosine kinases只會降低TNF-?釋放不會影響ROS產生,此差異顯示cAMP訊息傳導與Src tyrosine kinases活化狀態在TLR訊息傳導中有其調控之專一性。
Type 4 cAMP-specific phosphodisterases (PDE4s), the enzymes that degrade the second messenger cAMP, play a critical role in regulation of intracellular cAMP concentration. It has been demonstrated that PDE4 isozymes are expressed at high levels in immune cells and PDE4 inhibitors exhibit anti-inflammatory effects. revious studies have shown that lipopolysaccharide (LPS) induced PDE4B expression and activity in macrophages, and this induction is associated with an increase in the production of TNF-?. This TNF-? responses was inhibited by the PDE4 inhibitor rolipram. Moreover, LPS stimulates an increase in src tyrosine kinase activity, while is involved in various function of macrophages. In this study, we observed that rolipram, db-cAMP, and the Src kinase inhibitor PP2 attenuate LPS-induced TNF-? production in Raw 264.7 cells. In PDE4B-deficient macrophages, LPS-induced TNF-? release was decreased and the level of the decrease was similar to that observed in the wild-type macrophages inhibited by rolipram or PP2, indicating the effects of PDE4B on the TNF-? release are mediated by regulating cAMP signaling pathway as well as src tyrosine kinase activity. Moreover, measurements of PDE enzymatic activity indicated that LPS-induced PDE4 activity in macrophages was not inhibited by PP2, ruling out the possibility that PP2 attenuated TNF-? response isn’t derived from the inhibition of PDE4 by PP2. We also found that LPS-induced ROS production in macrophages was inhibited by cAMP or PKA activation, but not by PP2. In Raw 264.7 cells, LPS did not alter Lyn-tyr396 phosphorylation while decreased Lyn-tyr507 phosphorylation. In addition, the effect of rolipram on Lyn tyrosine phosphorylation was not exhibited as predicted. Taken together, these findings showed that inhibition of PDE4B activity reduces LPS-induced TNF-? release and ROS production, but interupting Src tyrosine kinase only attenuates the TNF-? release. This difference indicates that cAMP signaling and Src tyrosine kinase activation have their unique and specific role in regulation of TLR signaling.
中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 v
縮寫檢所表 vi
第一章 緒論 1
1.1環狀核苷酸磷酸二酯酶 (PDE) 1
1.2第四家族環狀核苷酸磷酸二酯酶 (Type 4 PDE;PDE4) 與巨噬細胞的免疫調控 2
1.3 Toll-like receptor (TLR) 之訊息傳導 3
1.4反應性含氧物種 (ROS) 4
1.5 ROS與免疫反應 5
1.6 Src tyrosine kinases 6
第二章 研究目的 9
第三章 實驗材料與方法 10
3.1實驗材料 10
3.2 實驗方法 11
3.2.1巨噬細胞之培養與處理方法 11
3.2.1.1 Raw 264.7細胞之培養與處理 11
3.2.1.2小鼠巨噬細胞收取培養與處理 12
3.2.1.3 HEK 293細胞之培養 13
3.2.2酵素免疫分析法 (ELISA) 14
3.2.3 PDE活性之檢測 14
3.2.4細胞萃取液之收集 15
3.2.5蛋白質濃度測定 16
3.2.6電擊轉染作用 (Electroporation transfection) 16
3.2.7 ROS含量測定 17
3.2.8西方墨點分析 17
第四章 實驗結果 20
4.1 LPS刺激巨噬細胞之TNF-?釋放會被cAMP與PP2抑制 20
4.2剔除PDE4B基因會降低LPS刺激巨噬細胞之TNF-?釋放 20
4.3 PP2對LPS誘導的PDE4活性無抑制作用 21
4.4 LPS誘導巨噬細胞產生ROS 22
4.5 PDE抑制劑對LPS刺激巨噬細胞產生ROS的影響 22
4.6 Src tyrosine kinase對LPS誘導巨噬細胞產生ROS之影響 23
4.7 Rolipram對Src與Lyn tyrosine磷酸化之影響 23
第五章 討論 25
5.1巨噬細胞中LPS誘導TNF-?表現與Src tyrosine kinase之關係 25
5.2 Src tyrosine kinase與PDE調控訊息傳遞之關係 26
5.3 LPS誘導細胞表現ROS與PDE調控cAMP之訊息傳遞 28
5.4巨噬細胞中Src tyrosine kinase與ROS表現之關係 29
第六章 參考文獻 30
圖 36
附錄一 46
附錄二 47
附錄三 48
1.Theodore J. T., “ Phosphodiesterase isozymes” American Journal of Respiratory and Critical Care Medicine, Vol 157, pp 351 – 370, 1998
2.Macro Conti and Joseph Beavo, “ Biochemistry and physiology of cyclic nucleotide phosphodiesterases: Essential components in cyclic nucleotide signaling “, Annual Review of Biochemistry, Vol 76, pp 481 – 511, March 2007
3.Miles D. Houslay, et al, “ Phosphodiesterase-4 as a therapeutic target “, Drug Discovery Today, Vol 10, pp 1503 – 1519, November, 2005
4.Jin S.-L. C., et al, “Induction of the cyclic nucleotide phosphodiesterase PDE4B is essential for LPS-activated TNF-? responses”, Proceedings of the National Academy of Sciences, Vol 99, pp7628 – 7633, May 2002
5.Jin S.-L. C., et al, “Specific Role of Phosphodiesterase 4B in lipopolysaccharide-induced signaling in mouse macrophages”, The Journal of Immunology, Vol 175, pp1523 – 1531, May 2005
6.Mario Galgani, et al, “Cyclic AMP modulates the functional plasticity of immature dendritic cells by inhibiting Src-like kinases through protein kinase A-mediated signaling”, The Journal of Biological Chemistry, Vol 279, pp32507 – 32514, July 2004
7.Gesine Hansen, et al, “ Absence of muscarinic cholinergic airway responses in mice deficient in the cyclic nucleotide phosphodiesterase PDE4D ”, Proceedings of the National Academy of Sciences, Vol 97, pp 6751 – 6756, March 2000
8.Yang Xiang, et al, “ Phosphodiesterase 4D is required for ?2 adrenoceptor subtype-specific signaling in cardiac myocytes “, Proceedings of the National Academy of Sciences, Vol 102, pp 909 – 914, January 2005
9.Miyako Ariga, et al, “ Nonredundant function of phosphodiesterases 4D and 4B in neutrophil recruitment to the site of inflammation “, The Journal of Immunology, Vol 173, pp 7531 – 7538, October 2004
10.Daniel Peter, et al, “ Differential expression and function of phosphodiesterase 4 (PDE4) subtypes in human primary CD4+T Cells: predominant role of PDE4D “, The Journal of Immunology, Vol 178, pp 4820 – 4831, January 2007
11.Frederic Geissmann, et al, “ Development of monocytes, macrophages, and dendritic Cells “, Science, Vol 327, pp 656 – 661, February 2010
12.Sudhanshu Bhushan, et al, “ Testicular innate immune defense against bacteria “ Molecular and Cellular Endocrinology, Vol 306, pp 37 – 44, 2009
13.Masayuki Fukata, et al, “ Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders”, Seminars in Immunology, Vol 21, pp 242 – 253, 2009
14.David D. Chaplin, “ Overview of the immune response “, The Journal of allergy and clinical immunology, Vol 125, pp 3 – 23, 2010
15.Mark A. Giembyze, “ Phosphodiesterase 4 inhibitoes and the treatment of asthma “, Drugs, Vol 59, pp 193 – 212, February 2000
16.C. Schudt, et al, “ PDE isoenzymes as targets for anti-asthma drugs “, pp 1179 – 1183, August 1995
17.Carlos G. Leon, et al, “ Discovery and development of Toll-Like receptor 4 (TLR4) antagonists: A new paradign for treating sepsis and other diseases “, Pharmaceutical Research, Vol 25, pp 1751 – 1761, August 2008
18.Taro Kawai and Shizuo Akira, “ Signaling to NF-kB by Toll-like receptors “, TRENDS in Molecular Medicine, Vol 13, pp 460 – 469, October 2007
19.Patti C. Zeidler, et al, “ Responses alveolar macrophages from inducible nitric oxide synthase knockout or wild-type mice to an in vitro lipopolysaccaride or silica expose “, Journal of Toxicology and Environmental Health, Vol 66, pp 995 – 1013, 2003
20.Maureen R. Gwinn, et al, “ Respiratory burst: Role in signal transduction in alveolar macrophages “, Journal of Toxicology and Environmental Health, Vol 9, pp 27- 39, January 2006
21.Geoffrey Gloire, et al, “ NF-kB activation by reactive oxygen spesis: Fifteen years later “, Biochemical Pharmacology, Vol 72, pp 1493 – 1505, 2006
22.Ji-Hee Kim, et al, “ The non-provitamin A carotenoid, lutein, Inhibits NF-kB-dependent gene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-kB-inducing kinase pathways: Role of H2O2 in NF-kB activation “, Free Radical Biology and Medicine, pp 1 – 12, 2008
23.Hye Sun Park, et al, “ TLR4 with NAD(P)H oxidase 4 isozymes is essential for lipopolysaccharide-induced production of reactive oxygen spesis and activation of NF-{kappa} B “, The Journal of Immunology, Vol 173, pp 3589 – 3593, 2004
24.Qiang Li, et al, “ Nox2 and Rac1 regulate H2O2-dependent recruitment of TRAF6 to endosomal interleukin-1 receptor complexes “, Molucular and Cellular Biology, Vol 26, pp 140 – 154, January 2006
25.JP Lu, et al, Prostate Cancer and Prostatic Diseases (2010)
26.Zhuang J., et al, “NADPH oxidase 4 mediates reactive oxygen species induction of CD146 dimerization in VEGF signal transduction “, Free Radical Biology and Medicine, Vol 49, pp 227 - 236, April 2010
27.D.M. Brown, et al, “ The effect of oxidative stress on macrophages and lung epithelial cells: The role of phosphodiesterases 1 and 4 “, Toxicology Letters, Vol 168, pp 1 – 6, 2007
28.Luay Y. Al-Essa, et al, “ Heterogeneity of circulating and exudated polymorphonuclear leukocytes in superoxide-generating response to cyclic AMP and cyclic AMP-elevating agents : Investigation of the underlying mechanism “, Biochemical Pharmacology, Vol 49, pp 315 – 322, January, 1995
29.Daisuke Okutani, et al, “ Src protein tyrosine family and acute inflammatory responses “, AJP-Lung Cellular and Molecular Physiology, Vol 291, pp 129 – 141, March 2006
30.Maria J. Smolinska, et al, “ Chemical inhibition of Src family kinases affects major LPS-activated pathways in primary human macrophages ”, Molecular Immunology, Vol 45, pp990 – 1000, September 2008
31.K. Tasken and A.J. Stokka, “ The molecular machinery for cAMP-dependent immunomodulation in T-cells “, Biochemical Society Transactions, Vol 34, pp 476 – 479, 2006
32.Irena Stefanova, et al, “ Lipopolysaccharide induces activation of CD14-associated protein tyrosine kinase p53/56lyn “, The Journal of Biological Chemistry, Vol 268, pp 20725 – 20728, 1994
33.Patricia Herrera-Velit, et al, “ Bacterial Lipopolysaccharide Induces the Association and Coordinate Activation of p53/56lyn and phosphatidylinositol 3-Kinase in Human Monocytes “, The Journal of Immunology, Vol 156, pp 1157 – 1165, 1996
34.Yuh-Ping Chong, et al, “ Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases” Biochimica and Biophysica Acta, Vol 1754, pp 210 – 220, September 2005
35.Thomas Schindler, et al, “ Crystal structure of Hck in complex with a Src family–selective tyrosine kinase inhibitor “, Molecular Cell, Vol 3, pp 639 – 648, May 1999
36.Katherine M. Knapp, et al, “ Ceramide-mediated stimulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in murine macrophages requires tyrosine kinase activity “ Journal of Leukocyte Biology, Vol 67, pp 735 – 741, May 2000
37.Fanying Meng, et al, “ Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn “, The Journal of Experimental Medicine, Vol 185, pp 1661 – 1670, May 1997
38.Dorit Avni, et al, “The ceramide-1-phosphate analogue PCERA-1 modulates tumour necrosis factor-a and interleukin-10 production in macrophages via the cAMP–PKA–CREB pathway in a GTP-dependent manner “, British Society for Immunology, Vol 129, pp 375 – 385, 2009
39.Dorit Avni, et al, “Role of CREB in modulation of TNF-? and IL-10 expression in LPS-stimulated RAW264.7 macrophages”, Molecular Immunology, Vol 47, pp1396 – 1403, February 2010
40.Leila Gobejishvili, et al, “Enhanced PDE4B expression augments LPS-inducible TNF expression in ethanol-primed monocytes: relevance to alcoholic liver disease”, AJP-Gastrointestinal and Liver Physiology, Vol 295, pp718 – 724, August 2008
41.Tzeng-Horng Leu, et al, “Lipopolysaccharide-induced c-Src expression plays a role in nitric oxide and TNF-? secretion in macrophages “, Molecular Immunology, Vol 43, pp308 – 316, May 2006
42.Xiaoni Kong, et al, “ NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2-deficient mice “, The Journal of Immunology, Vol 185, pp 569 – 577, May 2010
43.Shih-Chi Su, et al, “ LTA and LPS mediated activation of protein kinase in the regulation of inflammatory cytokines expression in macrophages “, Clinica Chimica Acta, Vol 374, pp 106 – 115, June 2006
44.Robert Roskoski Jr., “ Src protein-tyrosine kinase structure and regulation “, Biochemical and Biophysical Research Communications, Vol 324, pp 1155 – 1164, October 2004
45.Evan Ingley, “ Src family kinase: regulation of their activities, levels and identification of new pathways”, Biochimica and Biophysica Acta, Vol 1784, pp 56 – 65, 2008
46.Irens Stefanova, et al, “ Lipopolysaccaride induces activation of CD14-associated protein tyrosine kinase p53/56lyn “, The Journal of Biological Chemistry, Vol 268, pp 20725 – 20728, October, 1993
47.Jennifer Ckeck, et al, “vSrc kinase participates in LPS-induced activation of NADPH oxidase “, Molecular Immunology, Vol 47, pp 756 – 762, 2010
48.Simone Keck, et al, “ Activation of Murine Macrophages via TLR2 and TLR4 is negatively regulated by a Lyn/PI3K module and promoted by SHIP1 “, The Journal of Immunology, Vol 184, pp 5809 – 5818, 2010
49.Daisuke Aki, et al, “ Modulation of TLR signaling by the C-terminal Src kinase (Csk) in macrophages “, Genes to Cells, Vol 10, pp 357 – 368, 2005
50.Keith R.F. Elliott and Edward J. Leonard, “ Interaction of formylmethionyl-leucyl-phenylalanine, adenosine, and phosphodisterase inhibitors in human monocytes ”, FEBS Letters, Vol 254, pp 94 – 98, August 1989
51.K.H. Banner, et al, “ The effect of selective phosphodiesterase 3 and 4 isoenzyme inhibitors and established anti-asthma drugs on inflammatory cell activation “, British Journal of Pharmacology, Vol 119, pp 1255 – 1261, 1996
52.Naoya Murata, et al, “ Inhibition of superoxide anion production by extracellular acidification in neutrophils “, Cellular Immunology, Vol 259, pp 21 – 26, 2009
53.Ashis K. et al, “ Src-mediated tyrosine phosphorylation of p47phox in hyperxia-induced activation of NAPDH oxidase and generation od reactive oxygen species in lung endothelial cells “, The Journal of Biological Chemistry, Vol 280, pp 20700 – 20711, May 2005
54.Quan-Sheng Zhu, et al, “ G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth “, Blood, Vol 107, pp 1847 – 1856, March 2006
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top