臺灣博碩士論文加值系統

一氧化氮 (nitric oxide，NO)為一簡單分子，可經由一氧化氮合成酶(nitric oxide synthase，NOS)作用而產生。腎臟絲球體細胞(glomerular mesangial cells，MES-13 cells)經由lipopolysaccharide (LPS)和interferon-γ (IFN-γ)的處理，可活化誘發型一氧化氮合成酶(inducible NOS，iNOS)，進而產生大量的NO。在本篇研究論文中，探討cytochalasin D (微絲阻斷劑)、β,β’-Iminodipropionitrile (IDPN，中間絲阻斷劑)和nocodazole (微管阻斷劑)對LPS/IFN-γ刺激所產生之NO的影響。在不同濃度的cytochalasin D及nocodazole處理下，MES-13細胞在型態上及細胞存活率有明顯的變化，但在IDPN的處理下卻無影響。實驗結果亦顯示，cytochalasin D的處理並不會對NO產量或是iNOS蛋白質有影響；而在nocodazole處理下，細胞存活率、NO產量及iNOS蛋白質皆明顯減少；此外，MES-13細胞處理IDPN後，細胞存活率並無明顯下降，但NO產量、iNOS mRNA和蛋白質表現有隨著IDPN濃度增加而減少的趨勢。進一步實驗結果顯示IDPN可減弱ERK 1/2 MAPK之活化，以及降低NF-κB轉移至細胞核中。因此，我們認為IDPN是藉由抑制NF-κB和ERK的訊息傳遞路徑而抑制iNOS基因的表現及降低NO的產量。

Nitric oxide (NO) is a simple molecule that produced by nitric oxide synthase (NOS). Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulate the activation of inducible nitric oxide synthase (iNOS) and produce large amount of NO. In this study, the effect of cytochalasin D (an actin filament disrupting agent) , β,β’-Iminodipropionitrile (IDPN, an intermediate filaments disrupting agent), nocodazole (a microtubule disrupting agent) on LPS and IFN-γ-stimulated NO production in MES-13 cells were investigated. At different concentrations tested, the cell morphology and cell viability of LPS/ IFN-γ- treated MES-13 cells were dramatically altered after the treatment of cytochalasin D or nocodazole but not IDPN. NO production and iNOS protein expression showed no significant difference when treated with cytochalasin D. Nocodazole treatment reduced cell viabiblity, NO production and iNOS protein expression. Without affecting cell viability of MES-13 cells, IDPN decresed NO production, iNOS protein expression, and iNOS mRNA expression. IDPN decreased iNOS gene expressions through attenuated ERK1/2 MAPK activation, but had no effect on the p38 and JNK MAPKs, and attenuated nuclear fraction translocation of NF-κB. Thus, our data indicate that IDPN block LPS/IFN-γ- stimulated iNOS expression via inhibition of the ERK and NF-κB signaling pathways in MES-13 cells.

致謝 i
中文摘要 I
英文摘要 II
縮寫表 III
簡介 1
一、一氧化氮(Nitric oxide, NO) 1
二、一氧化氮合成酶(Nitric oxide synthase, NOS) 2
三、iNOS之調控 2
四、細胞骨架 (cytoskeletons) 4
五、細胞骨架阻斷劑 5
六、細胞骨架調控NOS 6
七、腎臟絲球體細胞 7
研究動機 8
實驗材料 9
藥品及試劑 9
抗體 10
實驗方法 11
細胞培養 (cell culture) 11
藥物處理 12
細胞存活率 (cell viability) 12
一氧化氮測定 12
樣品製備及蛋白濃度測定 13
核苷酸萃取 (RNA isolation) 14
RT-PCR (reverse transcription-polymerase chain reaction) 14
Real time PCR 16
質核分離 17
西方點墨法 (Western Blot) 18
EMSA (electrophoretic mobility shift assay) 19
免疫螢光顯微鏡 (immunofluorescent microscopy) 21
核苷酸干擾 (RNA interference) 22
吞噬作用實驗 (Phagocytosis assay) 22
結果 24
一、細胞骨架阻斷劑單獨處理MES-13對細胞存活率的影響 24
二、細胞骨架阻斷劑對LPS/IFN-γ刺激MES-13細胞產生之NO的影響 25
三、細胞骨架阻斷劑對LPS/IFN-γ刺激MES-13細胞誘發之iNOS蛋白質的影響 26
四、細胞骨架阻斷劑對LPS/IFN-γ刺激MES-13細胞型態的影響 27
五、IDPN對iNOS基因表現的影響 28
六、IDPN對轉錄因子的影響 29
七、IDPN對轉錄因子結合到iNOS基因啟動子區域之DNA序列的能力影響 31
八、降低中間絲蛋白質表現量是否會影響iNOS蛋白質表現 31
九、IDPN影響MES-13細胞吞噬作用之能力 32
討論 34
圖表 40
附圖 67
參考文獻 70

1.Aktan, F., iNOS-mediated nitric oxide production and its regulation. Life Sci, 2004. 75(6): p. 639-53.
2.Bogdan, C., Nitric oxide and the immune response. Nat Immunol, 2001. 2(10): p. 907-16.
3.Singh, S. and A.K. Gupta, Nitric oxide: role in tumour biology and iNOS/NO-based anticancer therapies. Cancer Chemother Pharmacol, 2011. 67(6): p. 1211-24.
4.Illi, B., et al., NO sparks off chromatin: tales of a multifaceted epigenetic regulator. Pharmacol Ther, 2009. 123(3): p. 344-52.
5.Fuseler, J.W. and M.T. Valarmathi, Modulation of the migration and differentiation potential of adult bone marrow stromal stem cells by nitric oxide. Biomaterials, 2012. 33(4): p. 1032-43.
6.Pautz, A., et al., Regulation of the expression of inducible nitric oxide synthase. Nitric Oxide, 2010. 23(2): p. 75-93.
7.Petrov, T., et al., Upregulation of iNOS expression and phosphorylation of eIF-2alpha are paralleled by suppression of protein synthesis in rat hypothalamus in a closed head trauma model. J Neurotrauma, 2001. 18(8): p. 799-812.
8.Geoffrey M. Cooper, R.E.H., The Cell: A Molecular Approach, 5th Edition.
9.Kumar, N., et al., Requirement of vimentin filament assembly for beta3-adrenergic receptor activation of ERK MAP kinase and lipolysis. J Biol Chem, 2007. 282(12): p. 9244-50.
10.May, J.A., et al., GPIIb-IIIa antagonists cause rapid disaggregation of platelets pre-treated with cytochalasin D. Evidence that the stability of platelet aggregates depends on normal cytoskeletal assembly. Platelets, 1998. 9(3-4): p. 227-32.
11.Cadet, J.L., The iminodipropionitrile (IDPN)-induced dyskinetic syndrome: behavioral and biochemical pharmacology. Neurosci Biobehav Rev, 1989. 13(1): p. 39-45.
12.Takahashi, N. and B. Ishizuka, The involvement of neurofilament heavy chain phosphorylation in the maturation and degeneration of rat oocytes. Endocrinology, 2012. 153(4): p. 1990-8.
13.Takahashi, N., W. Tarumi, and B. Ishizuka, Acute reproductive toxicity of 3,3''-iminodipropionitrile in female rats. Reprod Toxicol, 2012. 33(1): p. 27-34.
14.Galigniana, M.D., et al., Heat shock protein 90-dependent (geldanamycin-inhibited) movement of the glucocorticoid receptor through the cytoplasm to the nucleus requires intact cytoskeleton. Mol Endocrinol, 1998. 12(12): p. 1903-13.
15.Feuilloley, M., et al., Effect of the intermediate filament inhibitor IDPN on steroid secretion by frog adrenal glands. J Steroid Biochem, 1988. 30(1-6): p. 465-7.
16.Durham, H.D., The effect of beta,beta''-iminodipropionitrile (IDPN) on cytoskeletal organization in cultured human skin fibroblasts. Cell Biol Int Rep, 1986. 10(8): p. 599-610.
17.Su, Y., D. Kondrikov, and E.R. Block, Cytoskeletal regulation of nitric oxide synthase. Cell Biochem Biophys, 2005. 43(3): p. 439-49.
18.Takemoto, M., et al., Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation, 2002. 106(1): p. 57-62.
19.Witteck, A., et al., Rho protein-mediated changes in the structure of the actin cytoskeleton regulate human inducible NO synthase gene expression. Exp Cell Res, 2003. 287(1): p. 106-15.
20.Su, Y., S.I. Zharikov, and E.R. Block, Microtubule-active agents modify nitric oxide production in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol, 2002. 282(6): p. L1183-9.
21.Kagami, S. and S. Kondo, Beta1-integrins and glomerular injury. J Med Invest, 2004. 51(1-2): p. 1-13.
22.Schreiner, G.F., The mesangial phagocyte and its regulation of contractile cell biology. J Am Soc Nephrol, 1992. 2(10 Suppl): p. S74-82.
23.Ben-Ze''ev, A., Animal cell shape changes and gene expression. Bioessays, 1991. 13(5): p. 207-12.
24.Helfand, B.T., et al., Intermediate filament proteins participate in signal transduction. Trends Cell Biol, 2005. 15(11): p. 568-70.
25.Murakami, M., et al., Cellular components that functionally interact with signaling phospholipase A(2)s. Biochim Biophys Acta, 2000. 1488(1-2): p. 159-66.
26.Meriane, M., et al., Cdc42Hs and Rac1 GTPases induce the collapse of the vimentin intermediate filament network. J Biol Chem, 2000. 275(42): p. 33046-52.
27.Caulin, C., et al., Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis. J Cell Biol, 2000. 149(1): p. 17-22.
28.Johnson, R.J., et al., Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation. J Clin Invest, 1991. 87(3): p. 847-58.
29.Muchir, A., W. Wu, and H.J. Worman, Reduced expression of A-type lamins and emerin activates extracellular signal-regulated kinase in cultured cells. Biochim Biophys Acta, 2009. 1792(1): p. 75-81.
30.Webb, J.L., et al., Macrophage nitric oxide synthase associates with cortical actin but is not recruited to phagosomes. Infect Immun, 2001. 69(10): p. 6391-400.
31.Zeng, C. and A.R. Morrison, Disruption of the actin cytoskeleton regulates cytokine-induced iNOS expression. Am J Physiol Cell Physiol, 2001. 281(3): p. C932-40.
32.Marczin, N., et al., Cytoskeleton-dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells. Br J Pharmacol, 1996. 118(5): p. 1085-94.
33.Marczin, N., et al., Prevention of nitric oxide synthase induction in vascular smooth muscle cells by microtubule depolymerizing agents. Br J Pharmacol, 1993. 109(3): p. 603-5.
34.Liu, B.H., et al., The fungal metabolite, citrinin, inhibits lipopolysaccharide/interferon-gamma-induced nitric oxide production in glomerular mesangial cells. Int Immunopharmacol, 2010. 10(12): p. 1608-15.
35.Tsai, K.D., et al., Differential effects of LY294002 and wortmannin on inducible nitric oxide synthase expression in glomerular mesangial cells. Int Immunopharmacol, 2012. 12(3): p. 471-80.
36.Whitmarsh, A.J. and R.J. Davis, Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med (Berl), 1996. 74(10): p. 589-607.
37.Tegeder, I., J. Pfeilschifter, and G. Geisslinger, Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J, 2001. 15(12): p. 2057-72.
38.Bertelli, E., et al., Nestin expression in adult and developing human kidney. J Histochem Cytochem, 2007. 55(4): p. 411-21.
39.Miller, K.J. and H.A. Gonzalez, Serotonin 5-HT2A receptor activation inhibits cytokine-stimulated inducible nitric oxide synthase in C6 glioma cells. Ann N Y Acad Sci, 1998. 861: p. 169-73.
40.Chae, H.S., et al., 5-hydroxytryptophan acts on the mitogen-activated protein kinase extracellular-signal regulated protein kinase pathway to modulate cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 cells. Biol Pharm Bull, 2009. 32(4): p. 553-7.
41.Fenteany, G. and M. Glogauer, Cytoskeletal remodeling in leukocyte function. Curr Opin Hematol, 2004. 11(1): p. 15-24.