跳到主要內容

臺灣博碩士論文加值系統

(18.97.9.172) 您好!臺灣時間:2025/02/11 12:55
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:李錦綉
研究生(外文):Jin-Shiou Li
論文名稱:Dexamethasone在PC12細胞株影響PACAP基因表現的研究
論文名稱(外文):Effect of Dexamethasone on the Gene Expression of PACAP in PC12 Cell Line
指導教授:鄭瑞棠鄭瑞棠引用關係
指導教授(外文):Juei-Tang Cheng
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:74
中文關鍵詞:兒茶酚胺糖質皮質固醇嗜鉻細胞神經星狀膠細胞
外文關鍵詞:dexamethasoneglucocorticoidPACAPPC12astrocytecatecholamine
相關次數:
  • 被引用被引用:0
  • 點閱點閱:185
  • 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
  Pituitary adenylate cyclase-activating polypeptide (PACAP)屬於一種神經肽 (neuropeptide)類物質, 存在於腎上腺及交感神經節,可以調節兒茶酚胺(catecholamine )的合成及釋放。研究指出,PACAP與糖質皮質固醇 (glucocorticoid )都能刺激腎上腺的嗜鉻細胞 (chromaffin cell)合成兒茶酚胺,而增加兒茶酚胺在嗜鉻細胞的含量。因此,在腎上腺嗜鉻細胞,糖質皮質固醇有可能藉由調控PACAP來增加兒茶酚胺的含量。於是,使用培養的大白鼠腎上腺嗜鉻細胞 (pheochromocytoma 12 cell; PC12 cell ),以dexamethasone (DEXA)處理後,利用RT-PCR檢測,發現PACAP的基因表現會被DEXA所活化;前處理糖質皮質固醇受體 (glucocorticoid receptor ; GR) 的阻斷劑RU486 (Mifepristone)能夠抑制這個作用。此外,利用actinomycin D阻斷轉錄活性,發現DEXA能夠透過活化轉錄活性來誘發PACAP基因表現。
  另一方面,poly-ADP (ribose) polymerease (PARP)為一種分佈於細胞核內的酵素。研究指出,PARP對於核內蛋白的轉錄活性也扮演著重要的角色。因此,想去探討PARP酵素是否參與在GR活化後所影響的PACAP基因表現。於是,在處理DEXA後,將細胞分別投予兩種不同的PARP抑制劑: 3-aminobenzamide (3-AB)及1,5-isoquinolinediol (ISO),發現都能夠降低DEXA誘發的PACAP基因表現。接著,將細胞與DEXA共同培養24小時,利用HPLC測PC12細胞內的Dopamine (DA)含量,發現DEXA會增加細胞內DA含量的作用;且會被PACAP第一型受體(PAC1)的抑制劑PACAP (6-38)所阻斷,證實DEXA會透過調控PACAP來增加兒茶酚胺的合成。因此,可以推測 : 在PC12細胞株,DEXA能夠活化GR來提升PACAP的基因表現,進而增加了細胞內的兒茶酚胺含量。
  β-amyloid (Aβ)是一種具有神經毒性的peptide,在阿茲海默症患者的腦內可發現Aβ所凝聚而成的斑塊(plaque)。研究已證實,Aβ在神經細胞的堆積會引起細胞的凋零和死亡。另ㄧ方面,在神經細胞,PACAP是一種促進細胞存活的神經滋養因子。本實驗發現Aβ[1-42](1μM)會抑制PC12及第一型大白鼠腦部神經星狀膠細胞 ( rat brain astrocyte type 1;RBA1)的PACAP表現。接著,將PC12細胞處理DEXA之後,Aβ抑制PACAP的作用則會被DEXA逆轉。而且,DEXA的作用也會被GR的抑制劑RU486所阻斷。
  文獻指出,17β-estradiol (E2)和人參皂苷都具有神經保護的功能。在RBA1細胞,本研究也看到E2和人參皂苷Rh2會刺激PACAP基因表現,也能夠逆轉Aβ對PACAP的抑制作用。因此,在PC12細胞,DEXA會藉由活化GR來增加PACAP的表現,減少Aβ對PACAP基因的抑制作用。在RBA1細胞,E2和Rh2也可能藉由刺激PACAP的表現,來降低Aβ對神經細胞所產生的毒性。
 Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide, is observed in the adrenal gland and sympathetic ganglia to regulate catecholamine (CA) synthesis and release. Both of PACAP and glucocorticoid have been reported to elevate CA content in the line from stimulating biosynthesis. We assumed that glucocorticoid could modulate PACAP gene expression to increase CA content in adrenal chromaffin cell. In this study, we found that PACAP mRNA in rat pheochromocytoma cell (PC12 cell) examined by RT-PCR was stimulated after treatment of dexamethasone (DEXA). This effect was blocked by pre-incubation with RU486(Mifepristone), a glucocorticoid receptor(GR) antagonist. In addition, transcriptional inhibitor actinomycin D was used to confirm that DEXA could elevate PACAP gene expression through activating its transcriptional activity.
 Poly (ADP -ribose ) polymerase (PARP), an enzyme that exists in the nucleus, has been reported to play an important role in regulating the transcription of particular sets of genes. Thus, we investigated the role of PARP on GR modulating PACAP gene. PC12 cells were treated with two different PARP inhibitors, 3-aminobenzamide (3-AB) and 1,5- isoquinolinediol (ISO). The effect of DEXA on PACAP gene were inhibited after treatment of 3-AB or ISO, respectively.
 Dopamine (DA) content of PC12 cells was measured with HPLC after incubation for 24h with DEXA. DEXA increases DA content of PC12 cells and this effect could be blocked by pre-incubation with PACAP type 1 (PAC1) receptor antagonist PACAP(6-38). This result indicates that DEXA could increase CA content in PC12 cells via modulating PACAP gene. Therefore, we proved that DEXA has the ability to stimulate PACAP gene transcription via activating GR in PC12 cells, resulted in the increase of CA content.
 β-amyloid(Aβ) is a neurotoxic peptide, deposits of Aβ peptide are found in plaques in the brains with Alzheimer’s disease (AD). It has been reported that the intracellular and extracellular deposits of Aβ may lead to neuronal cell death and apoptosis. In neuron cell, PACAP is a neurotropic factor, which could promote cell survival. In this study, we found that incubation of PC12 or rat brain astrocyte type 1 (RBA1) with Aβ (1μM) could inhibit the endogenous PACAP expression. The inhibition effect of Aβ could be rescued by DEXA, and the effect of DEXA was blocked by RU486.
 It has been reported that both 17β-estradiol (E2) and ginseng extract – gensenoside Rh2 have neuroprotective effect. In this experiment, treatment of RBA1 cells with E2 or Rh2 could stimulate PACAP gene expression, respectively. And both of them could reverse Aβ reduced PACAP gene. Thus, our results revealed that, in PC12 cells, DEXA could reverse Aβ reduced PACAP gene via activating GR. In RBA1 cells, E2 and Rh2 could attenuate Aβ induced toxicity via reversing Aβ reduced PACAP gene expression.
縮寫表                  2
第一章 緒論               4
第二章 實驗方法及材料          13
第三章 實驗結果             25
第四章 討論               34
第五章 結論               40
參考文獻                 42
附圖                   50
自述                   74
Aloe L, Levi-Montalcini R. Nerve growth factor-induced transformationof immature chromaffin cells in vivo into sympathetic neurons: effect of anti-sense to nerve growth factor. Proc Natl Acad Sci USA 76 :1246-1250 (1979).
Arimura A. Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept 37 :287-303 (1992).
Arimura A. Somogyvari-Vigh A. Weill C. Fiore RC. Tatsuno I. Bay V. Brenneman DE. PACAP functions as a neurotrophic factor. Ann NY Acad Sci 739 :228–243 (1994).
Arimura A, Shioda S.Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors: neuroendocrine and endocrine interaction. Front Neuroendocrinol 16 :53– 88 (1995).
Behl C, Widmann M, Trapp T and Holsboer F 17-beta estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochem Biophys Res Commun 216 :473-482 (1995).
Behl C, Skutella T, Lezoualc’h F, Post A, Widmann M, Newton CJ, Holsboer F. Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol Pharmacol 51 :535–541 (1997).
Christophe J. Type I receptors for PACAP (a neuropeptide even more important than VIP?). Biochim Biophys Acta 1154 :183-199 (1993).
Corbitt J, Vivekananda J, Wang SS , Strong R . Transcriptional and Posttranscriptional Control of Tyrosine Hydroxylase Gene Expression During Persistent Stimulation of Pituitary Adenylate Cyclase- Activating Polypeptide Receptors on PC12 Cells: Regulation by Protein Kinase A-Dependent and Protein Kinase A-Independent Pathways. J Neurochem 71 :478-486 (1998).
D’Amours D, Desnoyers S, D’Silva I, Poirier GG. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem J 42 :249–268 (1999).
De Bosscher K, Vanden Berghe W , Haegeman G. The Interplay between the Glucocorticoid Receptor and Nuclear Factor-κB or Activator Protein-1: Molecular Mechanisms for Gene Repression. Endocrine Reviews 24 :488–522 (2003).
Deutsch P J and Sun Y. The 38 amino acid form of pituitary adenylate cyclase activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neunte outgrowth. J Biol Chem 267 : 5108-5113 (1992).
Einer-Jensen N, Carter AM. Local transfer of hormones between blood vessels within the adrenal gland may explain the functional interaction between the adrenal cortex and medulla. Med Hypotheses 44 :471-474 (1995).
Fitzpatrick JL, Mize AL, Wade CB, Harris JA, Shapiro RA, Dorsa DM Estrogen-mediated neuroprotection against β-amyloid toxicity requires expression of estrogen receptor α or β and activation of the MAPK pathway. J Neurochem 82 :674–682 (2002).
Ghatei MA, Takahashi K, Suzuki Y, Gardiner J, Jones PM, Bloom SR. Distribution, molecular characterization of pituitary adenylate cyclase activating polypeptide and its precursor encoding messenger RNA in human and rat tissues. J Endocrinol 136 :159 –166 (1993).
Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73 :2424 –2428 (1976).
Ha HC, Hester LD, and Snyder SH. Poly(ADP-ribose) polymerase-1 dependence of stress-induced transcription factors and associated gene expression in glia. Proc Natl Acad Sci USA 99 : 3270-3275 (2002).
Harrington WR, Sheng S, Barnett DH, Petz LN, Katzenellenbogen JA, Katzenellenbogen BS. Activities of estrogen receptor alpha- and beta-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression. Mol Cell Endocrinol 206 :13-22 (2003).
Hodel A. Effects of Glucocorticoids on Adrenal Chromaffin Cells. J Neuroendocrinol 13 :217-221 (2001)..
Isobe K, Nakai T, Takuwa Y. Ca(2+ ) -dependent stimulatory effect of pituitary adenylate cyclase-activating polypeptide on catecholamine secretion from cultured porcine adrenal medullary chromaffin cells. Endocrinology 132 :1757-1765 (1993).
Jacques D, Yu LS, Michel C, Yves G, Andre DL, Mona N , Thomas JS. Novel glucocorticoid receptor complex with DNA element of the hormone- repressed POMC gene. EMBO 12 :145-156 (1993).
Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, Multhaup, G, Beyreuther, K , Mueller-Hill, B. The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 325 :733–736 (1987).
Kawas C, Resnick S, Morrison A, Brookmeyer R, Corrada M, Zonderman A, Bacal C, Lingle DD, Metter E. A prospective study of estrogen replacement therapy and the risk of developing Alzheimer's disease: the Baltimore Longitudinal Study of Aging. Neurology 48 :1517-1521 (1997).
Kim H, Bang OY, Jung MW, Ha SD, Hong HS, Huh K, Kim SU, Mook-Jung I. Neuroprotective effects of estrogen against β-amyloid toxicity are mediated by estrogen receptors in cultured neuronal cells. Neurosci Lett 302 :58–62 (2001).
Kim KT, Park DH, Joh TH. Parallel up-regulation of catecholamine biosynthetic enzymes by dexamethasone in PC12 cells. J Neurochem 60 :946–951 (1993).
Kim YC, Kim SR, Markelonis GJ, Oh TH. Ginsenosides Rb1 and Rg3 protect cultured rat cortical cells from glutamate-induced neurodegeneration. J Neurosci Res 4 :426-432 (1998).
Kraus WL, Lis JT. PARP goes transcription. Cell 113 :677-683 (2003).

Liao B, Newmark H, Zhou R. Neuroprotective effects of ginseng total saponin and ginsenosides Rb1 and Rg1 on spinal cord neurons in vitro. Exp Neurol 173 :224-234 (2002).
Lowry DH, Rosenbrough NJ, Farr AI , Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 193 :265-275 (1951).
Liu IM, Chen WC, Cheng JT. Mediation of beta-endorphin by isoferulic acid to lower plasma glucose in streptozotocin-induced diabetic rats. J Pharmacol Exp Ther 307 :1196-204 (2003).
Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD,Coy DH. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164 :567–574 (1989).
Müller TH, Unsicker K. Nerve growth factor and dexamethasone modulate synthesis and storage of catecholamines in cultured rat adrenal medullary cells: dependence on postnatal age. J Neurochem 46 : 516-524 (1986).
Onoue S, Endo K, Ohshima K, Yajima T , Kashimoto K. The neuropeptide PACAP attenuates β-amyloid (1–42)-induced toxicity in PC12 cells. Peptides 23 :1471–1478 (2002).
Kantor O, Heinzlmann A, Suzuki N, Vincze E, Kocsis K, Koves K. Distribution of PACAP and its mRNA in several nonneural tissues of rats demonstrated by sandwich enzyme immunoassay and RT-PCR technique. Regul Pept 109 :103-105 (2002).
Powolny A, Takahashi K, Hopkins R G , Loo G. Induction of GADD Gene Expression by Phenethylisothiocyanate in Human Colon Adenocarcinoma Cells. J Cell Biochem 90 :1128– 1139 (2003).
Laburthe M, Couvineau A, Marie JC. VPAC receptors for VIP and PACAP. Receptors Channels 8 :137-153 (2002).
Rius RA, Guidotti A, Costa E. Pituitary adenylate cyclase activating polypeptide (PACAP) potently enhances tyrosine hydroxylase (TH) in adrenal chromaffin cells. Life Sci 54: 1735-1743 (1994).
Shintania N, Suetakea S, Hashimotoa H, Kogaa K, Kasaia A, Kawaguchia C, Moritaa Y, Hirosea M, Sakaia Y, Tomimotoa S, Matsudab T, Baba A. Neuroprotective action of endogenous PACAP in cultured rat cortical neurons. Regulatory Peptides 126 :123– 128 (2005).
Stachowiak MK, Hong JS, Viveros OH. Coordinate and differential regulation of phenylethanolamine N-methyltransferase, tyrosine hydroxylase and proenkephalin mRNA by neural and hormonal mechanisms in cultured bovine bovine adrenal medullary cells. Brain Res 510 :277-288 (1990).
Sugaya A, Yuzurihara M, Tsuda T, Yasuda K, Kajiwara K, Sugaya AE. Proliferative effect of ginseng saponin on neurite extension of primary cultured neurons of the rat cerebral cortex. J Ethnopharmaco 22 :173-181 (1988).
Tackikawa E, Kudo K, Harada K, Kashimoto T, Miyate M, Kakizaki A. Effects of ginseng saponins on responses induced by various receptor stimuli. Eur J Pharmacol 369 :23-32 (1999).
Tischler AS, Perlman RL, Nunnemacher G, Morse GM, DeLillis RA, Wolfe HJ, Sheard BE. Long-term effects of dexamethasone and nervegrowth factor on adrenal medullary cells cultured from young adult rats.Cell Tissue Res 225 :525-542 (1982).
Tischler AS, Perlman RL, Morse GM, Sheard BE. Glucocorticoids increase catecholamine synthesis and storage in PC12 pheochromocytoma cell cultures. J Neurochem 40 :364-370 (1983).
Troy CM, Rabacchi SA, Xu Z, Maroney AC, Connors TJ, Shelanski ML, Greene LA.β-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation. J Neurochem 77 :157-164 (2001).
Unsicker K, Krisch B, Otten U, Thoenen H. Nerve growth factor-induced fibre outgrowth from isolated rat adrenal chromaffin cells: impairment by glucocorticoids. Proc Natl Acad Sci USA 75 :3498-3502 (1978).
Vaudry D, Gonzalez BJ, Basille M, Pamantung TF, Fontaine M, Fournier A, Vaudry H. The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase- 3/CPP32. Proc Natl Acad Sci USA 97 :13390-13395 (2000).
Wakade AR, Wakade TD, Poosch M, Bannon MJ. Noradrenaline transport and transporter mRNA of rat chromaffin cells are controlled by dexamethasone and nerve growth factor. J Physiol 494 :67-75 (1996).
Wang JM, Tseng JT, Chang WC. Induction of human NF-IL6beta by epidermal growth factor is mediated through the p38 signaling pathway and cAMP response element-binding protein activation in A431 cells. Mol Biol Cell 16 :3365-3376 (2005).
Wen TC, Yoshimura H, Matsuda S, Lim JH, Sakanaka M. Ginseng root prevents learning disability and neuronal loss in gerbils with 5-minute forebrain ischaemia. Acta Neuropathol 91 :15-22 (1996).
Xu H, Gouras GK, Greenfield JP, Vincent B, Naslund J, Mazzarelli L, Fried G, Jovanovic JN, Seeger M, Relkin NR, Liao F, Checler F, Buxbaum JD, Chait BT, Thinakaran G, Sisodia SS, Wang R, Greengard P , Gandy S. Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides. Nature Med 4 :447-451 (1998).
Yan Z, Richard ML, Cynthia G , Andréa L. Selective cytotoxicity of intracellular β-amyloid peptide 1–42 through p53 and Bax in cultured primary human neurons. J Cell Biol 156 : 519–529 (2002).
Zhang Y & Rosenberg PA. Caspase-1 and poly (ADP-ribose) polymerase inhibitorsmay protect against peroxynitrite-induced neurotoxicity independent of their enzyme inhibitor activity. Eur J Neurosci 20 : 1727–1736 (2004).
Ame´ J-C, Jacobson EL & Jacobson MK. ADP-ribose polymer metabolism. In From DNA Damage and Stress Signalling to Cell Death: Poly ADP - Ribosylation Reactions. G de Murcia and S Shall, eds. (New York: Oxford University Press), pp. 1–34 (2002).
Nussey S & Whitehead S. Catecholamine synthesis and secretion. Endocrinology—An Integrated Approach. BIOS Scientific Publishers Ltd, London, UK (2001).
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top