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題名 親子分離對新生老鼠之腎上腺發育的影響
The effects of maternal separation on the development of adrenal glands
作者 周斈澧
貢獻者 賴桂珍
周斈澧
關鍵詞 嗜鉻細胞
親子分離
腎上腺素
正腎上腺素
日期 2011
上傳時間 3-Dec-2012 11:27:39 (UTC+8)
摘要 腎上腺素為哺乳類為了因應各種不同的壓力所釋放出的物質，影響生物體日常的生理功能且對生物體的存活有著莫大的貢獻，在成體中的調節已經在很多文獻中被探討，但對於處在發育階段的新生兒其腎上腺的發育與早期壓力對此系統的影響仍不盡清楚。為探討壓力與腎上腺發育及壓力調控之間的關係，本論文將觀察之重點放在腎上腺髓質中能夠製造腎上腺素的嗜鉻細胞(chromaffin cells)上。要了解新生兒腎上腺發育與壓力之間的關係，主要是利用親子分離的實驗，對新生老鼠造成壓力，實驗將新生老鼠分成三組，分別為控制組(control)、隔離組(isolate;P2~P14一小時/天)與撫摸組(handle;P2~P14 十分鐘/天)，試驗完畢後分別在老鼠出生後十四天(P14)與出生後二十一天(P21)進行腎上腺切片，利用腎上腺素合成酵素(PNMT ，phenylethanolamine-N-methyl transferase)及腎上腺素與正腎上腺素共同合成酵素(TH，tyrosine hydroxylase)之螢光免疫染色，來區分可製造腎上腺素的chromaffin cells，發現不管在P14或是P21，三組之間的腎上腺髓質結構並無太大差異。結果顯示，經過親子分離實驗的操弄，發現在撫摸組chromaffin cells中其PNMT的含量相對於TH的比例含量高於控制組與隔離組，顯示在撫摸組中每一chromaffin cell含有較多的PNMT，可能可以製造較多的腎上腺素。最後，為探討親子分離實驗對新生老鼠之腎上腺素與正腎上腺素含量的影響，以HPLC檢測經過親子分離實驗的新生老鼠之腎上腺，發現在P14時撫摸組與隔離組之腎上腺素含量比控制組高(p<0.05)，而正腎上腺素的含量則沒有差異;在P21時三組間的腎上腺素與正腎上腺素含量則沒有差異。而同樣的親子分離實驗在一胎一組新生老鼠的腎上腺發育上未看到任何顯著影響。
參考文獻 Alison J. WINDER (1991)New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase. Eur J. Biochcm. f98, 317-326
Andrej Tillinger (2010)Vesicular Monoamine Transporters (VMATs) in Adrenal Chromaffin Cells: Stress-Triggered Induction of VMAT2
and Expression in Epinephrine Synthesizing Cells .Cell Mol Neurobiol30:1459–1465
Baker PF ,Knight DE.(1978) Calcium-dependent exocytosis in bovine
adrenal medullary cells with leaky plasma membranes. Nature
276: 620–622.
Banks, P.(1965) Effects of stimulation by carbachol on the metabolism of the bovine adrenal medulla. Biochem. J.97: 555.
Baruchin A, Vollmer RR, Miner LL, Sell SL, Stricker EM,Kaplan BB.(1993) Cold-induced increases in phenylethanolamine Nmethyltransferase
(PNMT) mRNA are mediated by non-cholinergic mechanisms in the rat adrenal gland. Neurochem Res 18: 759–766.
Beatriz Galán-Rodríguez(2004)Extra-adrenal chromaffin cells of the Zuckerkandl´s paraganglion: morphological and electrophysiological study. Cell Biology of the Chromaffin Cell
Benedict J. Kolber (2008) HPA axis dysregulation and behavioral analysis of mouse mutants with altered GR or MR function. NIH Public Access 11(5): 321–338
Blaschko (I942)The activity OF I(-)-dopa decarboxylase J. Physiol. IOI, 337-
Blazicek P, Kvetnansky R(1989) Kinetic parameters of rat adrenal TH
and PNMT under acute and repeated stress. In: Stress: Neurochemical
and Humoral Mechanisms, edited by Van Loon GR, Kvetnansky
R, McCarty R, Axelrod J. New York: Gordon and Breach, p. 787–797
Benedict J. Kolber (2008) HPA axis dysregulation and behavioral analysis of mouse mutants with altered GR or MR function. NIH Public Access 11(5): 321–338
Cannon. (1926) Physiological regulation of normal states: some tentative postulates concerning biological homeostatics. p. 91. Paris: Éditions Médicales.
Chuang DM, Costa E.(1974) Biosynthesis of tyrosine hydroxylase in rat
adrenal medulla after exposure to cold. Proc Natl Acad Sci USA 71:
4570–4574.
Connett, R. J. ,Kirshner, N.(1970) Purification and properties of bovine phenylethanolamine N-methyltransferase.
J. Biol. Chem. 245: 329, 1970.
Coulter CL.(2004) Functional biology of the primate fetal adrenal gland: advances in technology provide new insight. Clinical and Experimental Pharmacology & Physiology.31: 475–484.
Coupland, R. E.(1965) Electron microscopic observations on the structure of the rat adrenal medulla. II. Normal innervation. J. Anat. 99: 255.
Cynthia G Zoski (2007) Handbook of electrochemistry P722
Douglas WW , Rubin RP(1961) The role of calcium in the secretory
response of the adrenal medulla to acetylcholine. J Physiol 159:
40–57.
Edith (1949) Formation of adrenaline from noradrenaline in
the perfused suprarenal gland. Brit. J. Pharmacol.,4, 245.
Ehrhart-Bornstein (1998) Intraadrenal Interactions in the Regulation of
Adrenocortical Steroidogenesis. Endocrine Reviews 19(2): 101–143
Erankii 0, Harkonen M (1963) Histochemical demonstration of fluorogenic amines in the cytoplasm of sympathetic ganglion cells of the rat. Acta Physiol Stand 58:285-286.
Fangwen Rao (2007) Tyrosine Hydroxylase, the Rate-Limiting Enzyme in Catecholamine Biosynthesis Circulation.116: 993-1006
Giuseppe Biagini (1998) Postnatal maternal separation during the stress hyporesponsive period enhances the adrenocortical response to novelty in adult rats by affecting feedback regulation in the CA1 hippocampal field. Neuroscience Volume 16, Issues 3–4 Pages 187–197
H. Winkler (1993) The adrenal chromaffin granule: a model for large dense core vesicles of endocrine and nervous tissue. J. Anat. 183, pp. 237-252
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描述 碩士
國立政治大學
神經科學研究所
97754003
100
資料來源 http://thesis.lib.nccu.edu.tw/record/#G0977540031
資料類型 thesis
dc.contributor.advisor 賴桂珍zh_TW
dc.contributor.author (Authors) 周斈澧zh_TW
dc.creator (作者) 周斈澧zh_TW
dc.date (日期) 2011en_US
dc.date.accessioned 3-Dec-2012 11:27:39 (UTC+8)-
dc.date.available 3-Dec-2012 11:27:39 (UTC+8)-
dc.date.issued (上傳時間) 3-Dec-2012 11:27:39 (UTC+8)-
dc.identifier (Other Identifiers) G0977540031en_US
dc.identifier.uri (URI) http://nccur.lib.nccu.edu.tw/handle/140.119/56332-
dc.description (描述) 碩士zh_TW
dc.description (描述) 國立政治大學zh_TW
dc.description (描述) 神經科學研究所zh_TW
dc.description (描述) 97754003zh_TW
dc.description (描述) 100zh_TW
dc.description.abstract (摘要) 腎上腺素為哺乳類為了因應各種不同的壓力所釋放出的物質，影響生物體日常的生理功能且對生物體的存活有著莫大的貢獻，在成體中的調節已經在很多文獻中被探討，但對於處在發育階段的新生兒其腎上腺的發育與早期壓力對此系統的影響仍不盡清楚。為探討壓力與腎上腺發育及壓力調控之間的關係，本論文將觀察之重點放在腎上腺髓質中能夠製造腎上腺素的嗜鉻細胞(chromaffin cells)上。要了解新生兒腎上腺發育與壓力之間的關係，主要是利用親子分離的實驗，對新生老鼠造成壓力，實驗將新生老鼠分成三組，分別為控制組(control)、隔離組(isolate;P2~P14一小時/天)與撫摸組(handle;P2~P14 十分鐘/天)，試驗完畢後分別在老鼠出生後十四天(P14)與出生後二十一天(P21)進行腎上腺切片，利用腎上腺素合成酵素(PNMT ，phenylethanolamine-N-methyl transferase)及腎上腺素與正腎上腺素共同合成酵素(TH，tyrosine hydroxylase)之螢光免疫染色，來區分可製造腎上腺素的chromaffin cells，發現不管在P14或是P21，三組之間的腎上腺髓質結構並無太大差異。結果顯示，經過親子分離實驗的操弄，發現在撫摸組chromaffin cells中其PNMT的含量相對於TH的比例含量高於控制組與隔離組，顯示在撫摸組中每一chromaffin cell含有較多的PNMT，可能可以製造較多的腎上腺素。最後，為探討親子分離實驗對新生老鼠之腎上腺素與正腎上腺素含量的影響，以HPLC檢測經過親子分離實驗的新生老鼠之腎上腺，發現在P14時撫摸組與隔離組之腎上腺素含量比控制組高(p<0.05)，而正腎上腺素的含量則沒有差異;在P21時三組間的腎上腺素與正腎上腺素含量則沒有差異。而同樣的親子分離實驗在一胎一組新生老鼠的腎上腺發育上未看到任何顯著影響。zh_TW
dc.description.tableofcontents 中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅲ
目錄……………………………………………………………………Ⅵ
圖次……………………………………………………………………Ⅹ
縮寫對照表……………………………………………………… XII
第一章、緒論………………………………………………………… 1
第一節 壓力的產生……………………………………………… 1
一、 壓力的定義………………………………………………… 1
二、 壓力的種類………………………………………………… 1
三、 壓力賀爾蒙的生理功能…………………………………… 2
第二節 壓力反應機制…………………………………………… 3
一、戰或逃反應(fight-or flight response）……………………. 3
二、hypothalamic-pituitary-adrenal axis(HPA axis) ………. 4
(1)HPA axis的組成 …………………………………………… 4
(2)HPA axis的反應機制 ……………………………………… 5
(3)HPA axis反應機制的調控 ………………………………… 5
(4)HPA axis對新生老鼠發育的影響 ………………………… 6
第三節 腎上腺發育過程………………………………………… 7
第四節 嗜鉻細胞(chromaffin cells)的生理功能…………… 8
一、嗜鉻細胞的介紹………………………………………… 8
二、腎上腺素與正腎上腺素的合成 …………………… 9
三、腎上腺素與正腎上腺素的儲存………………………… 11
四、腎上腺素與正腎上腺素的釋放………………………… 11
五、壓力與腎上腺素、正腎上腺素的關係………………… 12
第五節 與壓力賀爾蒙相關的疾病 ……………………… 13
第六節 與壓力相關的動物行為實驗 …………………… 15
一、親子分離(maternal separation) ………………… 15
二、高腳十字迷宮（Elevated Plus Maze、EPM） …… 15
三、其他與壓力相關的動物行為實驗……………………… 16
第七節 本論文之研究目的與策略 ……………………… 16
第二章、實驗材料與研究方法 …………………………… 18
第一節 實驗動物……………………………………………… 18
第二節 親子分離行為實驗(maternal separation) …………… 18
一、交配判定 ……………………………………………… 19
二、新生老鼠的標記 ……………………………………… 19
三、親子分離(一胎三組) …………………………………… 19
四、time course與親子分離(一胎一組) ………………… 20
第三節 初代細胞培養(primary cell culture) ………………20
一、 細胞培養皿之塗抹(coating plate) ………………………20
二、 培養基配製 ………………………………………………20
三、 初代細胞培養(primary cell culture) …………………21
第四節 免疫螢光染色(Immunofluorescence) …………………22
第五節 Chromaffin cell 的PNMT/TH強度(intensity)分析 …23
第六節 免疫組織化學染色(immunohistochemistry) …………23
一、組織切片的製備 ……………………………………………23
二、封片膠與明膠的製備 ………………………………………24
三、免疫螢光染色(Immunofluorescence) ……………………25
第七節 高壓液相層析儀檢測(HPLC; high performance liquid
chromatography) ………………………………………………26
一、移動相的配製 ………………………………………………26
二、standard的配製與catechoamine的萃取 ……………… 26
三、高壓液相層析儀(HPLC) ……………………………………27
第三章 結果 …………………………………………………………28
第一節 親子分離實驗對新生老鼠的腎上腺結構並無影響 ……28
第二節 親子分離實驗對新生老鼠其腎上腺可製造腎上腺素之chromaffin cells的影響 ………………………………………32
第三節 親子分離實驗影響新生老鼠之腎上腺素與正腎上腺素的含量 …………………………………………………………… 41
第四節 親子分離實驗(操弄至P21)對新生老鼠其腎上腺之腎上腺
素與正腎上腺素含量的影響………………………………… 49
第五節 親子分離實驗對一胎一組新生老鼠其腎上腺之腎上腺素與正腎上腺素含量的影響 …………………………………… 52
第四章 討論 ………………………………………………… 59
第五章 結論 ………………………………………………… 70
參考資料 ……………………………………………………… 71		zh_TW
dc.language.iso en_US-
dc.source.uri (資料來源) http://thesis.lib.nccu.edu.tw/record/#G0977540031en_US
dc.subject (關鍵詞) 嗜鉻細胞zh_TW
dc.subject (關鍵詞) 親子分離zh_TW
dc.subject (關鍵詞) 腎上腺素zh_TW
dc.subject (關鍵詞) 正腎上腺素zh_TW
dc.title (題名) 親子分離對新生老鼠之腎上腺發育的影響zh_TW
dc.title (題名) The effects of maternal separation on the development of adrenal glandsen_US
dc.type (資料類型) thesisen
dc.relation.reference (參考文獻) Alison J. WINDER (1991)New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase. Eur J. Biochcm. f98, 317-326
Andrej Tillinger (2010)Vesicular Monoamine Transporters (VMATs) in Adrenal Chromaffin Cells: Stress-Triggered Induction of VMAT2
and Expression in Epinephrine Synthesizing Cells .Cell Mol Neurobiol30:1459–1465
Baker PF ,Knight DE.(1978) Calcium-dependent exocytosis in bovine
adrenal medullary cells with leaky plasma membranes. Nature
276: 620–622.
Banks, P.(1965) Effects of stimulation by carbachol on the metabolism of the bovine adrenal medulla. Biochem. J.97: 555.
Baruchin A, Vollmer RR, Miner LL, Sell SL, Stricker EM,Kaplan BB.(1993) Cold-induced increases in phenylethanolamine Nmethyltransferase
(PNMT) mRNA are mediated by non-cholinergic mechanisms in the rat adrenal gland. Neurochem Res 18: 759–766.
Beatriz Galán-Rodríguez(2004)Extra-adrenal chromaffin cells of the Zuckerkandl´s paraganglion: morphological and electrophysiological study. Cell Biology of the Chromaffin Cell
Benedict J. Kolber (2008) HPA axis dysregulation and behavioral analysis of mouse mutants with altered GR or MR function. NIH Public Access 11(5): 321–338
Blaschko (I942)The activity OF I(-)-dopa decarboxylase J. Physiol. IOI, 337-
Blazicek P, Kvetnansky R(1989) Kinetic parameters of rat adrenal TH
and PNMT under acute and repeated stress. In: Stress: Neurochemical
and Humoral Mechanisms, edited by Van Loon GR, Kvetnansky
R, McCarty R, Axelrod J. New York: Gordon and Breach, p. 787–797
Benedict J. Kolber (2008) HPA axis dysregulation and behavioral analysis of mouse mutants with altered GR or MR function. NIH Public Access 11(5): 321–338
Cannon. (1926) Physiological regulation of normal states: some tentative postulates concerning biological homeostatics. p. 91. Paris: Éditions Médicales.
Chuang DM, Costa E.(1974) Biosynthesis of tyrosine hydroxylase in rat
adrenal medulla after exposure to cold. Proc Natl Acad Sci USA 71:
4570–4574.
Connett, R. J. ,Kirshner, N.(1970) Purification and properties of bovine phenylethanolamine N-methyltransferase.
J. Biol. Chem. 245: 329, 1970.
Coulter CL.(2004) Functional biology of the primate fetal adrenal gland: advances in technology provide new insight. Clinical and Experimental Pharmacology & Physiology.31: 475–484.
Coupland, R. E.(1965) Electron microscopic observations on the structure of the rat adrenal medulla. II. Normal innervation. J. Anat. 99: 255.
Cynthia G Zoski (2007) Handbook of electrochemistry P722
Douglas WW , Rubin RP(1961) The role of calcium in the secretory
response of the adrenal medulla to acetylcholine. J Physiol 159:
40–57.
Edith (1949) Formation of adrenaline from noradrenaline in
the perfused suprarenal gland. Brit. J. Pharmacol.,4, 245.
Ehrhart-Bornstein (1998) Intraadrenal Interactions in the Regulation of
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