(3.238.186.43) 您好!臺灣時間:2021/03/01 15:26
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:何思瑩
研究生(外文):Szu-Ying Ho
論文名稱:大花咸豐草植物化合物polyacetylenic glucoside藉由PKCα路徑來影響β細胞胰島素分泌及凋亡
論文名稱(外文):A polyacetylenic glucoside from Bidens pilosaenhances insulin secretion and prevents apoptosis on β-cells through regulation of PKCα pathway
指導教授:張力天
指導教授(外文):Lee-Tian Chang
口試委員:王之仰梁熾龍
口試日期:2011-05-27
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:52
中文關鍵詞:β cell apoptosiscalreticulininsulin secretionER stressPKCαRIN-m5F cellstranslocation
外文關鍵詞:β cell apoptosiscalreticulininsulin secretionER stressPKCαRIN-m5F cellstranslocation
相關次數:
  • 被引用被引用:0
  • 點閱點閱:206
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
摘要

第二型糖尿病為一慢性漸進性的疾病。第二型糖尿病患者常因胰島素分泌不足以及胰島素敏感性降低而造成高血糖病變。胰島細胞功能低下與胰島素分泌不足會造成內質網緊迫,而內質網緊迫又與長期高糖產生的氧化壓力相關。胰島素分泌除了由葡萄糖刺激之外,protein kinase Cα (PKCα) 的活性也具調節作用。而cytopiloyne,一種萃取自大花咸豐草的polyacetylenic glucoside,調控胞內鈣離子與diacylglycerol二酸甘油脂來影響下游PKCα活性,其包括促進PKCα的自體磷酸化及由細胞質轉位到細胞膜。利用PKCα siRNA以及PKCα抑制劑GF109203X確認cytopiloyne增加胰島素分泌是經由PKCα途徑。PKCα活化之後會去磷酸化許多受質蛋白質,除了PKC本身以外,calreticulin (CRT) 也是其中之一。calreticulin (CRT) 是一個位於內質網內膜用來調控鈣離子進出內質網的蛋白,進而排解內質網緊迫與減少細胞凋亡。cytopiloyne 可以增加RIN-m5Fβ細胞calreticulin的表現及促進PKCα磷酸化calreticulin,也降低高糖培養下RIN-m5F β細胞ROS (Reactive Oxygen Species) 的產生及改善內質網緊迫分子標記 (BiP、CHOP、XBP-1) 表現,進而抑制高糖下胰島細胞凋亡。所以我們推論cytopiloyne是藉由影響PKCα- CRT交互作用的途徑影響胰島素分泌及改善內質網緊迫下的β細胞凋亡。

Abstract

Type 2 diabetes mellitus has become an epidemic disease. Chronic hyperglycemia of type 2 diabetes patients showed β-cell dysfunction and insulin insensitivity. β-cell dysfunction may also induced by endoplasmic reticulum stress which is related with abnormal calcium homeostasis and hyperglycemia produced reactive oxygen species. Besides glucose-induced insulin secretion, insulin secretion can also be modulated by protein kinase C α activity. Cytopiloyne, a polyacetylenic glucoside from Bidens pilosa, increases two PKCα activators, calcium and diacylglycerol. Cytopiloyne enhances insulin secretion from a βcell which can be blocked by PKC inhibitor or siRNA of PKCα. Calreticulin is a target of PKCα phosphorylation and the major Ca2+ -binding protein of the endoplasmic reticulum lumen. Cytopiloyne can induce calreticulin expression and increase intracellular Ca2+ level on RIN-m5F βcells. We suggested that the interaction of PKCα and calreticulin improved β-cell apoptosis. Moreover, β-cell apoptosis was affect by causes including calcium modulator calreticulin deficiency, hyperglycemia and reactive oxygen species. Our study confirmed that cytopiloyne regulated insulin secretion and prevents apoptosis on β-cell through PKCα-mediated pathway.

目 次

中文摘要 i

英文摘要 ii

目次 iii

圖次 vi

第一章 緒言 ..............................................1

第二章 文獻探討...........................................2

第一節 第二型糖尿病成因.................................2

第二節 現今糖尿病藥物以及中草藥.........................3

第三節 胰島素功能.......................................4

第四節 胰島細胞分泌機制.................................4

一、nutrition-induced pathway.........................4

二、non-nutrition-induced pathway.....................4

一、Protein kinase C.............................4

(一) PKC 之分類.................................5

(二) PKC之結構..................................5

(三) PKC之活化機制..............................6

(四) PKC和胰島細胞之相關性......................6

第五節 胰島細胞內質網緊迫...............................6

一、未經褶疊蛋白反應 (UPR)............................7

二、鈣離子恆定........................................7

第三章 材料方法..........................................10

第一節 實驗材料 (Chemicals)............................10

第二節 細胞培養 (Cell culture).........................11

第三節 流式細胞儀 (Flow cytometry).....................12

第四節 酵素免疫分析法 (Enzyme-linked immunosorbent assay , ELISA)...........................................12

第五節 蛋白質萃取及西方墨點法 (Western blotting)................................................13

第六節 小干擾RNA (Small interfering RNA, siRNA).......15

第七節 薄膜色層分析 (Thin layer chromatography, TLC)...16

第八節 測細胞內鈣離子濃度 (Measurement of intracellular Ca2+)....................................................16

第九節 RNA萃取法及反轉錄聚合酶連鎖反應 (RT-PCR)........16

第十節 免疫組織化學染色 (Immunohistochemistry).........18

第四章 結果..............................................21

第五章 討論..............................................33

References...............................................38

附圖.....................................................45

縮寫對照表...............................................51


References

Ackermann AM, Gannon M. Molecular regulation of pancreatic beta-cell mass development, maintenance, and expansion. J Mol Endocrinol 2007;38:193-206.
Ahren B. Galanin increases cytoplasmic calcium in insulin-producing RINm5F cells by activating phospholipase C. Biochem Biophys Res Commun 1996;221:89-94.
Allagnat F, Christulia F, Ortis F, et al. Sustained production of spliced X-box binding protein 1 (XBP1) induces pancreatic beta cell dysfunction and apoptosis. Diabetologia 2010;53:1120-1130.
Araki E, Oyadomari S, Mori M. Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus. Exp Biol Med (Maywood) 2003;228:1213-1217.
Ashby MC, Tepikin AV. ER calcium and the functions of intracellular organelles. Semin Cell Dev Biol 2001;12:11-17.
Bachar E, Ariav Y, Cerasi E, et al. Neuronal nitric oxide synthase protects the pancreatic beta cell from glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis. Diabetologia 2010;53:2177-2187.
Bachar E, Ariav Y, Ketzinel-Gilad M, et al. Glucose amplifies fatty acid-induced endoplasmic reticulum stress in pancreatic beta-cells via activation of mTORC1. PLoS One 2009;4:e4954.
Bode HP, Goke B. Protein kinase C activates capacitative calcium entry in the insulin secreting cell line RINm5F. FEBS Lett 1994;339:307-311.
Boden G, Duan X, Homko C, et al. Increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals. Diabetes 2008;57:2438-2444.
Cardozo AK, Ortis F, Storling J, et al. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells. Diabetes 2005;54:452-461.
Caspersen C, Pedersen PS, Treiman M. The sarco/endoplasmic reticulum calcium-ATPase 2b is an endoplasmic reticulum stress-inducible protein. J Biol Chem 2000;275:22363-22372.
Chambers KT, Unverferth JA, Weber SM, et al. The role of nitric oxide and the unfolded protein response in cytokine-induced beta-cell death. Diabetes 2008;57:124-132.
Chang CL, Chang SL, Lee YM, et al. Cytopiloyne, a polyacetylenic glucoside, prevents type 1 diabetes in nonobese diabetic mice. J Immunol 2007;178:6984-6993.
Chen JC, Wu ML, Huang KC, et al. HMG-CoA reductase inhibitors activate the unfolded protein response and induce cytoprotective GRP78 expression. Cardiovasc Res 2008;80:138-150.
Chiang YM, Chang CL, Chang SL, et al. Cytopiloyne, a novel polyacetylenic glucoside from Bidens pilosa, functions as a T helper cell modulator. J Ethnopharmacol 2007;110:532-538.
Cho M, Park S, Gwak J, et al. Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular beta-catenin protein. Biochem Biophys Res Commun 2008;367:195-200.
Choi SE, Shin HC, Kim HE, et al. Involvement of Ca2+, CaMK II and PKA in EGb 761-induced insulin secretion in INS-1 cells. J Ethnopharmacol 2007;110:49-55.
Clark RA, Li SL, Pearson DW, et al. Regulation of calreticulin expression during induction of differentiation in human myeloid cells. Evidence for remodeling of the endoplasmic reticulum. J Biol Chem 2002;277:32369-32378.
Cnop M, Welsh N, Jonas JC, et al. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes 2005;54 Suppl 2:S97-107.
Coppolino MG, Dedhar S. Calreticulin. Int J Biochem Cell Biol 1998;30:553-558.
Corbett JA. Insulin biosynthesis: the IREny of it all. Cell Metab 2006;4:175-176.
Cristina Castaneda-Patlan M, Razo-Paredes R, Carrisoza-Gaytan R, et al. Protein kinase C is involved in the regulation of several calreticulin posttranslational modifications. Int J Biochem Cell Biol 2010;42:120-131.
Cunha DA, Hekerman P, Ladriere L, et al. Initiation and execution of lipotoxic ER stress in pancreatic beta-cells. J Cell Sci 2008;121:2308-2318.
Davies SP, Reddy H, Caivano M, et al. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000;351:95-105.
Donath MY, Ehses JA, Maedler K, et al. Mechanisms of beta-cell death in type 2 diabetes. Diabetes 2005;54 Suppl 2:S108-113.
Fonseca SG, Burcin M, Gromada J, et al. Endoplasmic reticulum stress in beta-cells and development of diabetes. Curr Opin Pharmacol 2009;9:763-770.
Fu HY, Minamino T, Tsukamoto O, et al. Overexpression of endoplasmic reticulum-resident chaperone attenuates cardiomyocyte death induced by proteasome inhibition. Cardiovasc Res 2008;79:600-610.
Ganesan S, Calle R, Zawalich K, et al. Glucose-induced translocation of protein kinase C in rat pancreatic islets. Proc Natl Acad Sci U S A 1990;87:9893-9897.
Grassi C, D''Ascenzo M, Valente A, et al. Ca2+ channel inhibition induced by nitric oxide in rat insulinoma RINm5F cells. Pflugers Arch 1999;437:241-247.
Harris TE, Persaud SJ, Jones PM. Atypical isoforms of pKc and insulin secretion from pancreatic beta-cells: evidence using Go 6976 and Ro 31-8220 as Pkc inhibitors. Biochem Biophys Res Commun 1996;227:672-676.
Holden NS, Squires PE, Kaur M, et al. Phorbol ester-stimulated NF-kappaB-dependent transcription: roles for isoforms of novel protein kinase C. Cell Signal 2008;20:1338-1348.
Hoyer-Hansen M, Bastholm L, Szyniarowski P, et al. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell 2007;25:193-205.
Hsu YJ, Lee TH, Chang CL, et al. Anti-hyperglycemic effects and mechanism of Bidens pilosa water extract. J Ethnopharmacol 2009;122:379-383.
Ikegami T, Krilov L, Meng J, et al. Decreased glucagon responsiveness by bile acids: a role for protein kinase Calpha and glucagon receptor phosphorylation. Endocrinology 2006;147:5294-5302.
Jones PM, Persaud SJ. Protein kinases, protein phosphorylation, and the regulation of insulin secretion from pancreatic beta-cells. Endocr Rev 1998;19:429-461.
Kasono K, Yasu T, Kakehashi A, et al. Nicorandil improves diabetes and rat islet beta-cell damage induced by streptozotocin in vivo and in vitro. Eur J Endocrinol 2004;151:277-285.
Kass GE, Orrenius S. Calcium signaling and cytotoxicity. Environ Health Perspect 1999;107 Suppl 1:25-35.
Kazi JU, Soh JW. Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA. Biochem Biophys Res Commun 2007;364:231-237.
Kim R, Emi M, Tanabe K, et al. Role of the unfolded protein response in cell death. Apoptosis 2006;11:5-13.
Kimura T, Imaishi K, Hagiwara Y, et al. ERp57 binds competitively to protein disulfide isomerase and calreticulin. Biochem Biophys Res Commun 2005;331:224-230.
Kitiphongspattana K, Khan TA, Ishii-Schrade K, et al. Protective role for nitric oxide during the endoplasmic reticulum stress response in pancreatic beta-cells. Am J Physiol Endocrinol Metab 2007;292:E1543-1554.
Lacabaratz-Porret C, Launay S, Corvazier E, et al. Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study. Biochem J 2000;350 Pt 3:723-734.
Lee IS, Hur EM, Suh BC, et al. Protein kinase A- and C-induced insulin release from Ca2+ -insensitive pools. Cell Signal 2003;15:529-537.
Lehtihet M, Welsh N, Berggren PO, et al. Glibenclamide inhibits islet carnitine palmitoyltransferase 1 activity, leading to PKC-dependent insulin exocytosis. Am J Physiol Endocrinol Metab 2003;285:E438-446.
Lipson KL, Fonseca SG, Ishigaki S, et al. Regulation of insulin biosynthesis in pancreatic beta cells by an endoplasmic reticulum-resident protein kinase IRE1. Cell Metab 2006;4:245-254.
Luciani DS, Gwiazda KS, Yang TL, et al. Roles of IP3R and RyR Ca2+ channels in endoplasmic reticulum stress and beta-cell death. Diabetes 2009;58:422-432.
Marchetti P, Bugliani M, Lupi R, et al. The endoplasmic reticulum in pancreatic beta cells of type 2 diabetes patients. Diabetologia 2007;50:2486-2494.
Melloul D. Role of NF-kappaB in beta-cell death. Biochem Soc Trans 2008;36:334-339.
Michalak M, Corbett EF, Mesaeli N, et al. Calreticulin: one protein, one gene, many functions. Biochem J 1999;344 Pt 2:281-292.
Michalak M, Groenendyk J, Szabo E, et al. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J 2009;417:651-666.
Michalak M, Milner RE, Burns K, et al. Calreticulin. Biochem J 1992;285 ( Pt 3):681-692.
Morash SC, Rose SD, Byers DM, et al. Overexpression of myristoylated alanine-rich C-kinase substrate enhances activation of phospholipase D by protein kinase C in SK-N-MC human neuroblastoma cells. Biochem J 1998;332 ( Pt 2):321-327.
Morishima N, Nakanishi K, Takenouchi H, et al. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem 2002;277:34287-34294.
Nawrocki ST, Carew JS, Dunner K, Jr., et al. Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinase and induces apoptosis via ER stress in human pancreatic cancer cells. Cancer Res 2005;65:11510-11519.
Nesher R, Anteby E, Yedovizky M, et al. Beta-cell protein kinases and the dynamics of the insulin response to glucose. Diabetes 2002;51 Suppl 1:S68-73.
Oyadomari S, Takeda K, Takiguchi M, et al. Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway. Proc Natl Acad Sci U S A 2001;98:10845-10850.
Petersen OH, Fedirko NV. Calcium signalling: store-operated channel found at last. Curr Biol 2001;11:R520-523.
Rendon-Huerta E, Mendoza-Hernandez G, Robles-Flores M. Characterization of calreticulin as a protein interacting with protein kinase C. Biochem J 1999;344 Pt 2:469-475.
Rhodes CJ. Type 2 diabetes-a matter of beta-cell life and death? Science 2005;307:380-384.
Ribeiro CM, Putney JW. Differential effects of protein kinase C activation on calcium storage and capacitative calcium entry in NIH 3T3 cells. J Biol Chem 1996;271:21522-21528.
Robertson RP, Harmon J, Tran PO, et al. Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes. Diabetes 2004;53 Suppl 1:S119-124.
Robertson RP, Harmon J, Tran PO, et al. Glucose toxicity in beta-cells: type 2 diabetes, good radicals gone bad, and the glutathione connection. Diabetes 2003;52:581-587.
Ron D, Hubbard SR. How IRE1 reacts to ER stress. Cell 2008;132:24-26.
Sameermahmood Z, Raji L, Saravanan T, et al. Gallic acid protects RINm5F beta-cells from glucolipotoxicity by its antiapoptotic and insulin-secretagogue actions. Phytother Res 2010;24 Suppl 1:S83-94.
Sanchez-Margalet V, Lucas M, Solano F, et al. Sensitivity of insulin-secreting RIN m5F cells to undergoing apoptosis by the protein kinase C inhibitor staurosporine. Exp Cell Res 1993;209:160-163.
Soh JW, Weinstein IB. Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes. J Biol Chem 2003;278:34709-34716.
Straub SG, Sharp GW. Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes Metab Res Rev 2002;18:451-463.
Stumvoll M, Goldstein BJ, van Haeften TW. Type 2 diabetes: principles of pathogenesis and therapy. Lancet 2005;365:1333-1346.
Takahashi H, Namiki H. Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors. Biochem J 2007;405:331-340.
Thastrup O, Cullen PJ, Drobak BK, et al. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A 1990;87:2466-2470.
Toullec D, Pianetti P, Coste H, et al. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 1991;266:15771-15781.
Wang Q, Brubaker PL. Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice. Diabetologia 2002;45:1263-1273.
Wu J, Kaufman RJ. From acute ER stress to physiological roles of the Unfolded Protein Response. Cell Death Differ 2006;13:374-384.
Yamauchi Y, Hayashi M, Abe-Dohmae S, et al. Apolipoprotein A-I activates protein kinase C alpha signaling to phosphorylate and stabilize ATP binding cassette transporter A1 for the high density lipoprotein assembly. J Biol Chem 2003;278:47890-47897.
Zawalich W, Brown C, Rasmussen H. Insulin secretion: combined effects of phorbol ester and A23187. Biochem Biophys Res Commun 1983;117:448-455.
Zhang K, Kaufman RJ. Signaling the unfolded protein response from the endoplasmic reticulum. J Biol Chem 2004;279:25935-25938.
Zhang K, Kaufman RJ. From endoplasmic-reticulum stress to the inflammatory response. Nature 2008;454:455-462.
Zhang L, Lai E, Teodoro T, et al. GRP78, but Not Protein-disulfide Isomerase, Partially Reverses Hyperglycemia-induced Inhibition of Insulin Synthesis and Secretion in Pancreatic {beta}-Cells. J Biol Chem 2009;284:5289-5298.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文
 
系統版面圖檔 系統版面圖檔