Please use this identifier to cite or link to this item:
標題: 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
Research on the membrane current and resistance of mouse oocytes with patch clamp and two-electrode voltage clamp method
作者: 周季呈
Chou, Chi-Cheng
關鍵字: single patch clamp;單極電流鉗;two-electrode voltage clamp;雙極電壓鉗制法
出版社: 化學工程學系所
引用: 1.Anant, B. P., “Interaction between capacitative Ca2+ influx and Ca2+-dependent Cl− currents in Xenopus oocytes.” Pflügers Archiv European Journal of Physiology, Vol 430, 954-963, (1995) 2.Almers, W., P. R. Stanfield and W. Stühmer “Lateral distribution of sodium and potassium channels in frog skeletal muscle: measurements with a patch-clamp technique” J Physiol, Vol 336, 261-284 (1983) 3.Beck, E. J. and C. Manuel, “Kv4 channels exhibit modulation of closed-state inactivation in inside-out patches.” Biophys J, Vol 81, 867-883, (2001) 4.Berridge, M. J., “Capacitative calcium entry” Biochem J., Vol 312, 1-11, (1995) 5.Choe, H. and H. Sackin, “Improved preparation of Xenopus oocytes for patch-clamp recording.” Pflügers Archiv European Journal of Physiology, Vol 433, 648-652, (1997) 6.Engelman, D. M., “Membranes are more mosaic than fluid” Nature, Vol 438, 578-580, (2005) 7.Fan, Z., N. Keiko and H. Masayasu, “Pinacidil activates the ATP-sensitive K+ channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes.” Pflügers Archiv European Journal of Physiology, Vol 415, 387-389, (1989) 8.Hamill, O. P., A. Marty, E. Neher, B. Sakmann and F. J. Sigworth, “Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.” Pflügers Archiv European Journal of Physiology, Vol 391, 85-100 (1981) 9.Hodgkin, A. L. and A. F. Huxley, “Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo.” J. Physiol, Vol 116, 449-72, (1952) 10.Hamill, O. P., J. W. Lane and D.W. J. McBride, “Amiloride: a molecular probe for mechanosensitive channels.” Trends Pharmacol Sci, Vol 13, 373-6, (1992) 11.Jan, C. R., J. R. Thomas, R. M. Anthony and J. A. George, “Alterations in calcium channel currents underlie defective insulin secretion in a Transgenic mouse.” The Journal of biological chemistry, Vol. 271, 15478-15485, (1996) 12.Kolajova2, 3, M. and J. M. Baltz3,4,5, “Volume-Regulated anion and organic osmolyte channels in mouse zygotes.” Biology of reproduction, Vol 60, 964-972, (1999) 13.Lory, P., F. A. Rassendren, S. Richard, F. Tiaho and J. Nargeot, “Characterization of voltage-dependent calcium channels expressed in Xenopus oocytes injected with mRNA from rat heart.” J. Physiol, Vol 429, 95-112, (1990) 14.MacKinnon, R., A. D. Declan, M. C. João, A. P. Richard, K. Anling, M. G.. Jacqueline, L. C. Steven, T. C. Brian, “The structure of the potassium channel: molecular basis of K+ conduction and selectivity”, Science, Vol 280, 69-77, (1998) 15.Mathias, R. T., I. S. Cohen and C. Oliva “Limitations of the whole cell patch clamp technique in the control of intracellular concentrations” Biophysical Journal,Vol 58, 759-770 (1990) 16.Martinac B., “Mechanosensitive ion channels : molecules of mechanotransduction.” Journal of Cell Science, Vol 117, 2449-2460, (2004) 17.Melodye, L. B. and J. M. William, “Changes in sodium , calcium and potassium currents during early embryonic development of the ascidian boltenia villosa.” J. physiol, Vol 393,619-634, (1987) 18.Neher, E. and B. Sakmann, “The patch clamp technique.” Sci Am., Vol 266(3), 44-51, (1992) 19.Neher, E., B. Sakmann and J. H. Steinbach, “The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes.” Pflügers Archiv European Journal of Physiology, Vol 375, 219-228, (1978) 20.Nelson, D. L. and M. M. Cox, “Principles of biochemistry.” W. H. Freeman and Company, (2005) 21.Niels, F., H. B. Robert and C. B. Jan “Whole cell patch clamp recording Performed on a planar glass chip.” Biophys J, Vol 82, 3056-3062, (2002) 22.Okada, Y., S. Oiki, A. Hazama and S. Morishima “Criteria for the Molecular Identification of the Volume-Sensitive Outwardly Rectifying Cl- Channel.” J. Gen. Physiol., Volume 112, 365-367, (1998) 23.Peng, M., H. Liuyu, X. Zijian, H. H. Wu and A. Amir, “Partial inhibition of Na+/K+ -ATPase by Ouabain induces the Ca2+ -dependent expressions of early-response genes Ca2+ in Cardiac Myocytes” The Journal Of Biological Chemistry, Vol 271, 10372-10378, (1996) 24.Sackin, H., “Stretch-activated ion channels” Kidney International, Vol 48, 1134-1147, (1995) 25.Strange, K., F. Emma and P. S. Jackson,“Swelling-activated organic osmolyte efflux: A new role for anion channels”, Kidney International, Vol 48, 994-1003, (1995) 26.Sakmann, B. and E. Neher, “Single-Channel Recording”, Plenum, (1983) 27.Sun, X. P., S. Supplisson, S. Torres, G. Sachs and E. 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. Lang, “Flufenamic acid, mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas” FEBS Lett, Vol 30, 79-82, (1990) 32.Yang, X. C. and F. Sachs, “Characterization of stretch-activated ion channels in Xenopus oocytes.” J Physiol, Vol 431, 103-122, (1990) 33.Zhang,Y. and O. P. Hamill, “On the discrepancy between whole-cell and membrane patch mechanosensitivity in Xenopus oocytes”, The Journal of Physiology, Vol 523, 101-115, (2000) 34.Yeagle, P. L., “Lipid regulation of cell membrane structure and function.” The FASEB Journal, Vol 3, 1833-1842 (1989)
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。

Cells 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.
其他識別: U0005-0908200715302500
Appears in Collections:化學工程學系所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.