請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47650
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳俊宏(Jiun-Hong Chen) | |
dc.contributor.author | Chin-Ching Huang | en |
dc.contributor.author | 黃欽敬 | zh_TW |
dc.date.accessioned | 2021-06-15T06:10:40Z | - |
dc.date.available | 2010-08-19 | |
dc.date.copyright | 2010-08-19 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-12 | |
dc.identifier.citation | 參考文獻(References)
Alberti KG, Zimmet PZ. (1998). Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med. Jul;15(7):539-53. Andrali SS, Sampley ML, Vanderford NL, Ozcan S. (2008). Glucose regulation of insulin gene expression in pancreatic beta-cells. Biochem J. Oct 1;415(1):1-10. Angenstein F, Evans AM, Settlage RE, Moran ST, Ling SC, Klintsova AY, Shabanowitz J, Hunt DF, Greenough WT. (2002). A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons. J Neurosci. Oct 15;22(20):8827-37. Baliga BS, Pronczuk AW, Munro HN. (1969). Mechanism of cycloheximide inhibition of protein synthesis in a cell-free system prepared from rat liver. J Biol Chem. Aug 25;244(16):4480-9. Besson A, Wilson TL, Yong VW. (2002). The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility. J Biol Chem. Jun 14;277(24):22073-84. Brownlee M, Cerami A. (1981). The biochemistry of the complications of diabetes mellitus. Annu Rev Biochem.;50:385-432. Bhadada SK, Sahay RK, Jyotsna VP, Agrawal JK (2001). Diabetic Neuropathy: Current Concepts. J Indian Acad Clin Med. Oct-Dec; 2(4), 305-318. Buensuceso CS, Woodside D, Huff JL, Plopper GE, O'Toole TE. (2001). The WD protein Rack1 mediates protein kinase C and integrin-dependent cell migration. J Cell Sci. May;114(Pt 9):1691-8. Carlino A, Toledo H, Vidal V, Redfield B, Strassman J, Abdel-Ghany M, Racker E, Weissbach H, Brot N. (1994). BiP is a substrate for src kinase in vitro. Biochem Biophys Res Commun. Jun 30;201(3):1548-53. Ceci M, Gaviraghi C, Gorrini C, Sala LA, Offenhäuser N, Marchisio PC, Biffo S. (2003). Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly. Nature. Dec 4;426(6966):579-84. Chang BY, Harte RA, Cartwright CA. (2002). RACK1: a novel substrate for the Src protein-tyrosine kinase. Oncogene. Oct 31;21(50):7619-29. Chang HC, Nathan DF, Lindquist S. (1997). In vivo analysis of the Hsp90 cochaperone Sti1 (p60). Mol Cell Biol. Jan;17(1):318-25. Chen WY, Yang YM, Chuang NN. (2002). Selective enhanced phosphorylation of shrimp beta-tubulin by PKC-delta with PEP(taxol), a synthetic peptide encoding the taxol binding region. J Exp Zool. Mar 1;292(4):376-83. Chen S, Spiegelberg BD, Lin F, Dell EJ, Hamm HE. (2004). Interaction of Gbetagamma with RACK1 and other WD40 repeat proteins. J Mol Cell Cardiol. Aug;37(2):399-406. Chong YP, Ia KK, Mulhern TD, Cheng HC. (2005). Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases. Biochim Biophys Acta. Dec 30;1754(1-2):210-20. Chu LY, Chen YH, Chuang NN. (2005). Dimerize RACK1 upon transformation with oncogenic ras. Biochem Biophys Res Commun. May 6;330(2):474-82. Coulombe PA, Omary MB. (2002). 'Hard' and 'soft' principles defining the structure, function and regulation of keratin intermediate filaments. Curr Opin Cell Biol. Feb;14(1):110-22. Coulombe PA, Wong P. (2004). Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds. Nat Cell Biol. Aug;6(8):699-706. Cox EA, Bennin D, Doan AT, O'Toole T, Huttenlocher A. (2003). RACK1 regulates integrin-mediated adhesion, protrusion, and chemotactic cell migration via its Src-binding site. Mol Biol Cell. Feb;14(2):658-69. Gaddini L, Villa M, Matteucci A, Mallozzi C, Petrucci TC, Di Stasi AM, Leo L, Malchiodi-Albedi F, Pricci F. (2009). Early effects of high glucose in retinal tissue cultures Renin-Angiotensin system-dependent and -independent signaling. Neurobiol Dis. Aug;35(2):278-85. Galarneau L, Loranger A, Gilbert S, Marceau N. (2007). Keratins modulate hepatic cell adhesion, size and G1/S transition. Exp Cell Res. Jan 1;313(1):179-94. Desgrosellier JS, Cheresh DA.( 2010). Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. Jan;10(1):9-22. Duby JJ, Campbell RK, Setter SM, White JR, Rasmussen KA. (2004). Diabetic neuropathy: an intensive review. Am J Health Syst Pharm. Jan 15;61(2):160-73 Dunon D, Piali L, Imhof BA. (1996). To stick or not to stick: the new leukocyte homing paradigm. Curr Opin Cell Biol. Oct;8(5):714-23. Fears, C. Y., Gladson, C. L., & Woods, A. (2006). Syndecan-2 is expressed in the microvasculature of gliomas and regulates angiogenic processes in microvascular endothelial cells. J Biol Chem, 281(21), 14533-14536. Frame MC, Fincham VJ, Carragher NO, Wyke JA. (2002). v-Src's hold over actin and cell adhesions. Nat Rev Mol Cell Biol. Apr;3(4):233-45. Geijsen, N., Spaargaren, M., Raaijmakers, J. A., Lammers, J. W., Koenderman, L., & Coffer, P. J. (1999). Association of RACK1 and PKCbeta with the common beta-chain of the IL-5/IL-3/GM-CSF receptor. Oncogene, 18(36), 5126-5130. Gilbert S, Loranger A, Marceau N. (2004). Keratins modulate c-Flip/extracellular signal-regulated kinase 1 and 2 antiapoptotic signaling in simple epithelial cells. Mol Cell Biol. Aug;24(16):7072-81. Gu LH, Coulombe PA. (2007). Keratin function in skin epithelia: a broadening palette with surprising shades. Curr Opin Cell Biol. Feb;19(1):13-23. Guo W, Giancotti FG. (2004). Integrin signalling during tumour progression. Nat Rev Mol Cell Biol. Oct;5(10):816-26. Hayes MJ, Moss SE. (2009). Annexin 2 has a dual role as regulator and effector of v-Src in cell transformation. J Biol Chem. Apr 10;284(15):10202-10. Heiska L, Carpén O. (2005). Src phosphorylates ezrin at tyrosine 477 and induces a phosphospecific association between ezrin and a kelch-repeat protein family member. J Biol Chem. Mar 18;280(11):10244-52. Herrmann H, Aebi U. (2000). Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr Opin Cell Biol. Feb;12(1):79-90. Herrmann H, Aebi U. (2004). Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular Scaffolds. Annu Rev Biochem.;73:749-89. Hogervorst F, Kuikman I, von dem Borne AE, Sonnenberg A. (1990). Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain. EMBO J. Mar;9(3):765-70. Hood JD, Cheresh DA. (2002). Role of integrins in cell invasion and migration. Nat Rev Cancer. Feb;2(2):91-100. Huang CC, Liu CH, Chuang NN. (2008). An enhanced association of RACK1 with Abl in cells transfected with oncogenic ras. Int J Biochem Cell Biol.;40(3):423-31. Huang Q, Sheibani N. (2008). High glucose promotes retinal endothelial cell migration through activation of Src, PI3K/Akt1/eNOS, and ERKs. Am J Physiol Cell Physiol. Dec;295(6):C1647-57. Humphries JD, Byron A, Humphries MJ. (2006). Integrin ligands at a glance. J Cell Sci. Oct 1;119(Pt 19):3901-3. Hynes RO. (2002). Integrins: bidirectional, allosteric signaling machines. Cell. Sep 20;110(6):673-87. Inada H, Izawa I, Nishizawa M, Fujita E, Kiyono T, Takahashi T, Momoi T, Inagaki M. (2001). Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J Cell Biol. Oct 29;155(3):415-26. Kheifets V, Bright R, Inagaki K, Schechtman D, Mochly-Rosen D. (2006). Protein kinase C delta (deltaPKC)-annexin V interaction: a required step in deltaPKC translocation and function. J Biol Chem. Aug 11;281(32):23218-26. Kiely PA, Leahy M, O'Gorman D, O'Connor R. (2005). RACK1-mediated integration of adhesion and insulin-like growth factor I (IGF-I) signaling and cell migration are defective in cells expressing an IGF-I receptor mutated at tyrosines 1250 and 1251. J Biol Chem. Mar 4;280(9):7624-33. Koehler, J. A., & Moran, M. F. (2001). RACK1, a protein kinase C scaffolding protein, interacts with the PH domain of p120GAP. Biochem Biophys Res Commun, 283(4), 888-895. Ku NO, Azhar S, Omary MB. (2002). Keratin 8 phosphorylation by p38 kinase regulates cellular keratin filament reorganization: modulation by a keratin 1-like disease causing mutation. J Biol Chem. Mar 29;277(13):10775-82. Laemmli UK. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. Aug 15;227(5259):680-5. Lane LC. (1978). A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal Biochem. Jun 1;86(2):655-64. Leonardi R, Zhang YM, Rock CO, Jackowski S. (2005). Coenzyme A: back in action. Prog Lipid Res. Mar-May;44(2-3):125-53. Li Y, Chang Y, Zhang L, Feng Q, Liu Z, Zhang Y, Zuo J, Meng Y, Fang F. (2005). High glucose upregulates pantothenate kinase 4 (PanK4) and thus affects M2-type pyruvate kinase (Pkm2). Mol Cell Biochem. Sep;277(1-2):117-25. Liao J, Omary MB. (1996). 14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor. J Cell Biol. Apr;133(2):345-57. Liao J, Price D, Omary MB. (1997). Association of glucose-regulated protein (grp78) with human keratin 8. FEBS Lett. Nov 17;417(3):316-20. Liliental, J., & Chang, D. D. (1998). Rack1, a receptor for activated protein kinase C, interacts with integrin beta subunit. J Biol Chem, 273(4), 2379-2383. Loreni, F., Iadevaia, V., Tino, E., Caldarola, S., & Amaldi, F. (2005). RACK1 mRNA translation is regulated via a rapamycin-sensitive pathway and coordinated with ribosomal protein synthesis. FEBS Lett, 579(25), 5517-5520. Maller JL. (2001).The elusive progesterone receptor in Xenopus oocytes. Proc Natl Acad Sci U S A. Jan 2;98(1):8-10. Mamidipudi, V., Chang, B. Y., Harte, R. A., Lee, K. C., & Cartwright, C. A. (2004). RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells. FEBS Lett, 567(2-3), 321-326. Martin GS. (2001). The hunting of the Src. Nat Rev Mol Cell Biol. Jun;2(6):467-75. Mazurek S, Grimm H, Boschek CB, Vaupel P, Eigenbrodt E. (2002). Pyruvate kinase type M2: a crossroad in the tumor metabolome. Br J Nutr. Jan;87 Suppl 1:S23-9. Mazzalupo S, Wong P, Martin P, Coulombe PA. (2003). Role for keratins 6 and 17 during wound closure in embryonic mouse skin. Dev Dyn. Feb;226(2):356-65. Mitra SK, Hanson DA, Schlaepfer DD. (2005). Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol. Jan;6(1):56-68. Mourton T, Hellberg CB, Burden-Gulley SM, Hinman J, Rhee A, Brady-Kalnay SM. (2001). The PTPmu protein-tyrosine phosphatase binds and recruits the scaffolding protein RACK1 to cell-cell contacts. J Biol Chem. May 4;276(18):14896-901. Núñez A, Franco A, Madrid M, Soto T, Vicente J, Gacto M, Cansado J. (2009). Role for RACK1 orthologue Cpc2 in the modulation of stress response in fission yeast. Mol Biol Cell. Sep;20(18):3996-4009. Pallari HM, Eriksson JE. (2006) Intermediate filaments as signaling platforms. Sci STKE. Dec 19;2006(366):pe53. Paramio JM, Jorcano JL. (2002). Beyond structure: do intermediate filaments modulate cell signalling? Bioessays. Sep;24(9):836-44. Pass JM, Zheng Y, Wead WB, Zhang J, Li RC, Bolli R, Ping P. (2001). PKCepsilon activation induces dichotomous cardiac phenotypes and modulates PKCepsilon-RACK interactions and RACK expression. Am J Physiol Heart Circ Physiol. Mar;280(3):H946-55. Post CB, Gaul BS, Eisenmesser EZ, Schneider ML. (1999). NMR structure of phospho-tyrosine signaling complexes. Med Res Rev. Jul;19(4):295-305. Quan GX, Krell PJ, Arif BM, Feng Q. (2006). Receptor of activated C kinase 1 (RACK1) is necessary for the 20-hydroxyecdysone-induced expression of the transcription factor CHR3 in the spruce budworm Choristoneura fumiferana. Insect Mol Biol. Feb;15(1):79-87. Reizes O, Goldberger O, Smith AC, Xu Z, Bernfield M, Bickel PE. (2006).Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase. Biochemistry. May 9;45(18):5703-11. Robertson RP, Harmon JS. (2006). Diabetes, glucose toxicity, and oxidative stress: A case of double jeopardy for the pancreatic islet beta cell. Free Radic Biol Med. Jul 15;41(2):177-84. Ron, D., Chen, C. H., Caldwell, J., Jamieson, L., Orr, E., & Mochly-Rosen, D. (1994). Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins. Proc Natl Acad Sci U S A, 91(3), 839-843. Sang N, Severino A, Russo P, Baldi A, Giordano A, Mileo AM, Paggi MG, De Luca A. (2001). RACK1 interacts with E1A and rescues E1A-induced yeast growth inhibition and mammalian cell apoptosis. J Biol Chem. Jul 20;276(29):27026-33. Santos, M., Paramio, J. M., Bravo, A., Ramirez, A . & Jorcano, J. L. (2002). The expression of keratin k10 in the basal layer of the epidermis inhibits cell proliferation and prevents skin tumorigenesis. J. Biol. Chem. 277, 19122–19130. Schägger H, von Jagow G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. Nov 1;166(2):368-79. Schechtman, D., & Mochly-Rosen, D. (2001). Adaptor proteins in protein kinase C-mediated signal transduction. Oncogene, 20(44), 6339-6347. Shariat-Madar Z, Schmaier AH. (1999). Kininogen-cytokeratin 1 interactions in endothelial cell biology. Trends Cardiovasc Med. Nov;9(8):238-44. Shariat-Madar Z, Mahdi F, Schmaier AH. (2002). Assembly and activation of the plasma kallikrein/kinin system: a new interpretation. Int Immunopharmacol. Dec;2(13-14):1841-9. Sklan EH, Podoly E, Soreq H. (2006). RACK1 has the nerve to act: structure meets function in the nervous system. Prog Neurobiol. Feb;78(2):117-34. Suckale J, Solimena M. (2008). Pancreas islets in metabolic signaling--focus on the beta-cell. Front Biosci. May 1;13:7156-71. Switala-Jelen K, Dabrowska K, Opolski A, Lipinska L, Nowaczyk M, Gorski A. The biological functions of beta3 integrins. (2004). Folia Biol (Praha).;50(5):143-52. Tamareille S, Mignen O, Capiod T, Rücker-Martin C, Feuvray D. (2006). High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells. Cell Calcium. Jan;39(1):47-55. Tamkun JW, DeSimone DW, Fonda D, Patel RS, Buck C, Horwitz AF, Hynes RO. (1986). Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. Jul 18;46(2):271-82. Thorn JM, Armstrong NA, Cantrell LA, Kay BK. (1999). Identification and characterisation of Xenopus moesin, a Src substrate in Xenopus laevis oocytes. Zygote. May;7(2):113-22. Thornton C, Tang KC, Phamluong K, Luong K, Vagts A, Nikanjam D, Yaka R, Ron D. (2004). Spatial and temporal regulation of RACK1 function and N-methyl-D-aspartate receptor activity through WD40 motif-mediated dimerization. J Biol Chem. Jul 23;279(30):31357-64. Widmaier E, Raff H, and Strang K (2007). Vander's Human Physiology: The Mechanisms of Body Function with ARIS. 11 edition. McGraw-Hill Science/Engineering/Math. Wozniak MA, Modzelewska K, Kwong L, Keely PJ. (2004) Focal adhesion regulation of cell behavior. Biochim Biophys Acta. Jul 5;1692(2-3):103-19 Won, M., Park, S. K., Hoe, K. L., Jang, Y. J., Chung, K. S., Kim, D. U., Kim, H. B., & Yoo, H. S. (2001). Rkp1/Cpc2, a fission yeast RACK1 homolog, is involved in actin cytoskeleton organization through protein kinase C, Pck2, signaling. Biochem Biophys Res Commun, 282(1), 10-15. Yarovaya GA, Blokhina TB, Neshkova EA. (2002). Contact system. New concepts on activation mechanisms and bioregulatory functions. Biochemistry (Mosc). Jan;67(1):13-24. Yarwood, S. J., Steele, M. R., Scotland, G., Houslay, M. D., & Bolger, G. B. (1999). The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem, 274(21),14909-14917. Yeatman TJ. (2004). A renaissance for SRC. Nat Rev Cancer. Jun;4(6):470-80. Yedovitzky M, Mochly-Rosen D, Johnson JA, Gray MO, Ron D, Abramovitch E, Cerasi E, Nesher R. (1997). Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic beta-cells. J Biol Chem. Jan 17;272(3):1417-20. Zeller CE, Parnell SC, Dohlman HG. (2007). The RACK1 ortholog Asc1 functions as a G-protein beta subunit coupled to glucose responsiveness in yeast. J Biol Chem. Aug 24;282(34):25168-76. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47650 | - |
dc.description.abstract | 血糖是提供體內細胞穩定養分的來源,因此血糖濃度受恆定機制的嚴格調控,當血糖濃度過高或過低,人體就會產生疾病。血糖失調產生的疾病中,影響最廣泛的例子是糖尿病(diabetes mellitus)。絕大多數的糖尿病患者皆死於併發症,糖尿病神經病變(diabetic neuropathy)是最常見的糖尿病併發症之一。高血糖會對神經細胞造成壓力(stress)導致糖尿病神經病變,高血糖對神經細胞產生的變異包括細胞遷移(migration)、細胞附著(adhesion)等細胞生理的改變,而影響細胞遷移和細胞附著最重要的構造是focal adhesion。由於integrin, Src, RACK是構成focal adhesion的蛋白質之一,而cytokeratin又能調節focal adhesion的功能,因此本實驗的目標是探討高濃度葡萄糖對integrin和cytokeratin的交互作用在神經細胞的影響。
本實驗以NMB7 neuroblastoma cell line為實驗對象。一開始以親和管柱捕捉和RACK1及Src有交互作用的蛋白質,質譜儀鑑定的結果發現integrin αV包含其中。接著以免疫螢光染色法證實了integrin αVβ3和focal adhesion kinase (FAK)共同組成了NMB7細胞的focal adhision。接著以Q-PCR的技術去檢測,證明K8/K18是NMB7唯一含有的cytokeratin。免疫沉澱法的結果指出integrin αVβ3及K8/K18的交互作用會隨葡萄糖濃度的增加而增加。本實驗親合管柱及質譜儀的結果除了捕捉到和葡萄糖代謝有關的蛋白質,也捕捉到了壓力反應(stress response)蛋白質,而這些壓力反應蛋白質和integrin αVβ3 complex或K8/K18有所關聯,因此在葡萄糖濃度變化下integrin αVβ3及K8/K18的交互作用或許是NMB7因應葡萄糖濃度變化所產生的壓力反應(stress response)。 | zh_TW |
dc.description.abstract | The blood sugar is the stable source of energy for body's cells, so the body's homeostatic mechanism keeps blood glucose levels within a narrow range. When the blood glucose level of humans is out of this range, the individual will get diseases. One of the prevalent diseases is diabetes mellitus. Most of the patients with diabetes mellitus are died from diabetic complications. Diabetic neuropathy is one of the common diabetic complications. High blood sugar can stress nerve cells to develop the diabetic neuropathy, and also can alter cell migration and adhesion that are dependent on the dynamicity of focal adhesion in cells. Focal adhesion is composed of integrin, Src, RACK, and other proteins, and is regulated by cytokeratins. Thus, the main goal of this study is to explore the interactions between integrin and cytokeratin under high glucose situation.
In this study, NMB7 neuroblastoma cell line was used as the cell model. At beginning, affinity column chromatography was used to find the proteins that interacted with RACK1 and Src. Based on the result, integrin αV was one of them through mass spectrometry. Then, the focal adhesion complex composed of integrin αVβ3 and FAK in NMB7 cells was detected by immunostaining colocalization studies. Though Q-PCR technology, K8/K18 pairings were the only cytokeratins expressed in NMB7. From the previous data, a few stress-responded proteins that interact with integrin αVβ3 complex or K8/K18 were identified. Furthermore, the interaction between integrin αVβ3 and K8/K18 dependent on glucose concentration was related to stress response. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:10:40Z (GMT). No. of bitstreams: 1 ntu-99-R95b41013-1.pdf: 9812625 bytes, checksum: 1de43330f7f744c99c6d637597c3c7f3 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目錄(Contents)
口試委員會審定書…………………………………………………... i 誌謝(Acknowledgment)……………………………………………. ii 摘要…………………………………………………………………. iv Abstract……………………………………………………………... v 目錄(Contents)……………………………………………………... vi 序言(Introduction)………………………………………………….. 1 Blood Sugar and Disorders…………………………………………………... 1 Focal Adhesion………………………………………………………………. 3 Integrin……………………………………………………………………….. 4 Src…….……………………………………………………………………… 6 RACK1………………………………………………………………………. 7 Cytokeratin……..……………………………………………………………. 8 Main Goal…………………………………………………………………... 10 材料與方法(Materials and Methods)…………………………….. 11 細胞培養基(Culture medium)……………………………………………... 11 細胞培養(Cell culture)………. ……………………………………………. 11 細胞膜的粗萃取…………………………………………………………… 11 Hitrap-PEPTaxol和-RACK1Y246F親和管柱的製備…………………………. 12 Hitrap-PEPTaxol和-RACK1Y246F親和管柱的操作…………………………. 12 液相等電點聚焦法(Liquid-phase isoelectric focusing)…………………… 13 Tricine-SDS-PAGE gel電泳分析………………………………………….. 13 西方點墨分析法(Western immunoblotting)………………………………. 14 Coomassie blue R-250染色法……………………………………………... 15 質譜儀蛋白質身份鑑定…………………………………………………… 15 免疫螢光染色(Immunofluorescence staining; IF)………………………… 16 引子的設計(Primer designing)…………………………………………….. 17 抽取總量RNA………………………………………………………………. 18 置備cDNA (First Strand cDNA Synthesis)………………………………... 18 即時聚合酶連鎖反應(Real-Time Polymerase chain reaction; Q-PCR)…... 18 免疫沉澱法(Immunoprecipitation; IP)…………………………………….. 19 蛋白質定量………………………………………………………………… 19 葡萄糖和cycloheximide的添加…………………………………………… 20 結果(Results)………………………………………………………. 21 以親合管柱和質譜儀偵測和RACK1及Src有交互作用的蛋白質……... 21 以免疫螢光染色法檢測NMB7細胞的focal adhesion complex………… 22 以Q-PCR檢測NMB7的cytokeratin family成員………………………… 23 葡萄糖濃度對mRNA表現量的影響…………………………………….. 24 葡萄糖濃度對integrin β3及cytokeratin交互作用的影響………………. 24 Cycloheximide對integrin β3及cytokeratin交互作用的影響……………. 25 討論(Discussion)…………………………………………………... 27 以親合管柱辨認Integrin αV………………………………………………. 27 在NMB7的focal adhesion complex……………………………………… 28 NMB7所含的cytokeratin family成員……………………………………. 29 葡萄糖濃度對mRNA的影響…………………………………………….. 30 葡萄糖濃度對蛋白質的影響……………………………………………... 31 未來展望…………………………………………………………………... 32 參考文獻(References)……………………………………………... 35 圖(Figures) ………………………………………………………… 44 【圖1】在NMB7中,用質譜儀分析和RACK1且和Src有交互作用的蛋白質…………………………………………………………………………... 44 【圖2】在NMB7中,用質譜儀分析和RACK1且和Src有交互作用的蛋白質…………………………………………………………………………... 47 【圖3】在NMB7,和RACK1且和Src有交互作用的蛋白質中,integrin αV是其中之一………………………………………………………………... 50 【圖4】在NMB7中,和integrin αV配對的integrin β蛋白鏈…………….. 55 【圖5】在NMB7中的focal adhesion complex…………………………… 57 【圖6】NMB7所表現的cytokeratin family成員………………………… 61 【圖7】不同葡萄糖濃度下,PanK2和PanK4的mRNA表現量…………. 62 【圖8】不同葡萄糖濃度下,integrin β1和integrin β3的mRNA表現量…. 63 【圖9】不同葡萄糖濃度下,cytokeratin-8和cytokeratin-18的mRNA表現量…………………………………………………………………………... 64 【圖10】不同葡萄糖濃度下,integrin β1、integrin β3和cytokeratin的蛋白質表現量……………………………………………………………………... 65 【圖11】不同葡萄糖濃度下,integrin β3和cytokeratin的交互作用…….. 67 【圖12】滲透壓對integrin β3和cytokeratin交互作用的影響…………… 69 【圖13】Cycloheximide對integrin β3和cytokeratin交互作用的影響…... 71 附錄(Supplementary information) ………………………………. 73 【附圖1】NMB7所表現的cytokeratin family成員……………………… 73 【附圖2】不同葡萄糖濃度下,PanK2和PanK4的mRNA表現量………. 80 【附圖3】不同葡萄糖濃度下,integrin β1和integrin β3的mRNA表現量. 81 【附圖4】不同葡萄糖濃度下,cytokeratin-8和cytokeratin-18的mRNA表現量………………………………………………………………………... 82 【附圖5】不同葡萄糖濃度下,integrin β1、integrin β3和cytokeratin的蛋白質表現量…………………………………………………………………... 83 【附圖6】不同葡萄糖濃度下,integrin β3和cytokeratin的交互作用…… 85 【附圖7】滲透壓對integrin β3和cytokeratin交互作用的影響………….. 87 【附圖8】Cycloheximide處理NMB7的最適濃度和時間………………. 89 【附圖9】Cycloheximide處理NMB7的最適濃度和時間………………. 92 | |
dc.language.iso | zh-TW | |
dc.title | 高濃度葡萄糖影響下,integrin β3和cytokeratin的交互作用 | zh_TW |
dc.title | High Glucose Effect on the Interaction between Integrin β3 and Cytokeratin | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李心予(Hsinyu Lee),黃偉邦(Wei-Pang Huang) | |
dc.subject.keyword | 葡萄糖,integrin,cytokeratin,RACK1,Src, | zh_TW |
dc.subject.keyword | glucose,integrin,cytokeratin,RACK1,Src, | en |
dc.relation.page | 97 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-13 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 動物學研究所 | zh_TW |
顯示於系所單位: | 動物學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-99-1.pdf 目前未授權公開取用 | 9.58 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。