Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3622
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dc.contributor蔡豐羽zh_TW
dc.contributor鄭文桐zh_TW
dc.contributor.advisor張厚謙zh_TW
dc.contributor.advisorHou-Chien Changen_US
dc.contributor.author周季呈zh_TW
dc.contributor.authorChou, Chi-Chengen_US
dc.contributor.other中興大學zh_TW
dc.date2008zh_TW
dc.date.accessioned2014-06-06T05:32:19Z-
dc.date.available2014-06-06T05:32:19Z-
dc.identifierU0005-0908200715302500zh_TW
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Mayer “Characterization of large-conductance chloride channels in rabbit colonic smooth muscle.” J Physiol, Vol 448, 355-382, (1992) 28.Tomaselli, G. F., E. Marban, and G. Yellen“Sodium channels from human brain RNA expressed in Xenopus oocytes. Basic electrophysiologic characteristics and their modification by diphenylhydantoin” J Clin Invest, Vol 1989, 1724-1732, (1989) 29.Ussing, H. H. and C. L. Voûte, “Some morphological aspects of active sodium ttransport* : The Epithelium of the Frog Skin” The Journal of Cell Biology, Vol 36, 625-638, (1968) 30.Vanmolkot, K.R., E. E. Kors, J. J. Hottenga, G. M. Terwindt, J. Haan, W. A. Hoefnagels, D. F. Black, L. A. Sandkuijl, R. R. Frants, M. D. Ferrari, A. M. van den Maagdenberg, “Novel mutations in the Na+, K+-ATPase pump gene ATP1A2 associated with familial hemiplegic migraine and benign familial infantile convulsions.” Annals of neurology, Vol 54, 360-6, (2003) 31.Gögelein*, H., D. Dahlem, H. C. Englert and H. J. 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dc.identifier.urihttp://hdl.handle.net/11455/3622-
dc.description.abstract細胞是所有生物體功能和構造的基礎單位,然而幾乎所有的類型種類的細胞都有其共通點就是細胞膜(plasma membrane) 的存在,其決定了細胞大小範圍、組織複雜反應的順序、作用在訊號接收及能量轉型。膜由大量的脂質與蛋白質分子組成。而流體鑲嵌模型敘述了細胞膜的基本構造。細胞膜對無機離子(k+、Na+、Cl-)和大部分的其他帶電荷或極性分子的自由通透具有阻斷的功能。細胞膜上的運輸蛋白(transport proteins),使某些離子和分子可以通透於細胞膜。其他的膜蛋白(membrane proteins)包含受體(receptors)和酵素皆為運送離子分子的機制。而膜的選擇性通透將可維持特定離子或分子在胞內的濃度分佈。各種細胞之細胞膜內所包含的離子通道種類各式各樣,正因為離子通道的獨特性存在,致使細胞能扮演該有的角色。然而研究對象皆是由受精卵分化後所形成的細胞,因為離子通道扮演重要的生理表現機制,所以就算在原始尚未受精的小鼠卵母細胞膜中,應該也佔有一定的份量存在。 本研究將以兩種不同的測量模式來研究卵母細胞膜的電生理表現以及氯離子通道在小鼠卵母細胞中所扮演的角色,第一種方法是利用單極電流鉗接細胞膜的方式,將細胞放置於充滿M2培養液的載玻片中;藉由改變玻璃電極內電導液(M2、KCl、NaCl、CaCl2)成分以及濃度,對細胞膜進行鉗制所得到之電流變化。添加氯離子通道抑制劑(NPPB) 於M2培養液後將對膜電流產生明顯的變化。第二種方法則是利用雙極電壓鉗制法(TEVC),利用透過電壓鉗將膜電位控制在一定的數值,在藉由電流鉗做全細胞回饋電流的接收。將以未加入細胞的M2培養液、加入M2內的卵母細胞、以及添加氯離子通道抑制劑的卵母細胞、鈉鉀幫浦抑制劑作為TEVC的實驗對象。其中可以充分的了解到膜電阻的大小並非一定值,而是隨著膜內離子通道數目的多寡、以及打開的數量而定。從添加過抑制劑的卵母細胞全電流可以看出膜電阻因為氯離子通道的阻塞造成阻力係數增加,也可知道鈉鉀幫浦只是一種運送離子的機制,其功能在抑制後並不會讓膜電阻增加,反而會使膜電阻降低。原因為Ouabain除了抑制鈉鉀幫浦外,還有另ㄧ項功能就是當幫浦失活後會啟動另ㄧ連鎖反應使鈣離子通道活化,讓膜電阻降低。zh_TW
dc.description.abstractCells are the structural and functional units of all living organisms. The plasma membrane defines the periphery of the cell, separating its contents from the surroundings. It is composed of lipid and protein molecules. Transport proteins in the plasma membrane allow the passage of certain ions(k+、Na+、Cl-) and molecules. The selected permeation of membrane maintains specific ion and molecule concentration inside and outside of the cell at desired levels. Ion channels play crucial roles in physiology and their functions are remarkable not only in differentiated cells but also the unfertilized cells like oocytes. This study uses two kinds of different measuring methods to study electrophysiological responses of the oocyte membrane. First method deals with using single patch clamp to record the membrane currents. In-cell profiles of ionic concentration are measured at various compositions of internal solution in the glass pipette. Experimental results find that adding the Cl- channel blocker(NPPB) causes the M2 medium currents change obviously. Potential mechanisms are proposed to interpret this phenomenon. Second method employs two-electrode voltage clamp(TEVC) with holding on specific membrane potential to record the whole cell membrane currents. And by adding the Cl- channel blocker and Na+-K+ pump inhibitor(Ouabain) it is found that the membrane resistances are not constant, but depend on the numbers of ion channel and their open probabilities.zh_TW
dc.description.tableofcontents摘要 I Abstract II 圖 目 錄 V 表 目 錄 VI 第一章 前言 1 第二章 文獻回顧 3 2-1細胞膜的構造 3 2-1.1 細胞膜的模型與結構 4 2-2 物質運送與訊息傳遞 6 2-2.1被動運輸 7 2-2.2主動運輸 8 2-3電生理技術發展 9 2-3.1電生理史 9 2-3.2生物電訊號紀錄 9 2-3.3電壓鉗、電流鉗,與膜片鉗 10 2-3.4 膜片鉗之電流與電導記錄 15 2-3.5 鈉、鉀、鈣離子通道特性與結構 16 2-3.6卵母細胞的各種電生理表現 17 2-3.7基礎電生理表現 20 第三章 實驗材料與方法 24 3-1 實驗目的 24 3-2 實驗流程 24 3-3 實驗藥品 25 3-4 實驗器材 26 3-5 實驗儀器 26 3-6 實驗步驟 27 第四章 實驗結果與探討 35 4-1 培養液背景電流測定 35 4-2 各種電極液之離子濃度狀態下測定背景電流變化 38 4-3 各種電極液之成份對卵母細胞膜電流變化之測定 40 4-3.1 卵母細胞的離子交換對M2培養液的基礎電流影響 42 4-3.2 離子通道抑制劑對卵母細胞膜電流之影響 42 4-4 利用 two electrode voltage clamp 紀錄M2背景電壓、電流變化 45 4-5 利用 two electrode voltage clamp 紀錄細胞膜表面電壓、電流變化 47 4-5.1 TEVC法,添加NPPB對膜電流的影響 48 4-5.2 TEVC法,添加 Ouabain 對膜電流的影響 49 第五章 結論與未來展望 51 第六章 參考文獻 52 附錄A 在TEVC法下,膜電容、放電電流表示圖 56 附錄B TEVC法調控電壓測量M2培養液回饋電流 57 附錄 C TEVC法調控電壓測量全細胞膜電流 60 附錄 D TEVC法在添加NPPB後調控電壓測量全細胞膜電流 63 附錄E TEVC法在添加Ouabain 30 μA後調控電壓測量全細胞膜電流 66 附錄F TEVC法在添加Ouabain 100 μA後調控電壓測量全細胞膜電流 69zh_TW
dc.language.isoen_USzh_TW
dc.publisher化學工程學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0908200715302500en_US
dc.subjectsingle patch clampen_US
dc.subject單極電流鉗zh_TW
dc.subjecttwo-electrode voltage clampen_US
dc.subject雙極電壓鉗制法zh_TW
dc.title使用膜片鉗與雙極電壓鉗法對小鼠卵母細胞之膜電流與膜電阻研究zh_TW
dc.titleResearch on the membrane current and resistance of mouse oocytes with patch clamp and two-electrode voltage clamp methoden_US
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.fulltextno fulltext-
item.languageiso639-1en_US-
item.grantfulltextnone-
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