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研究生:李傳德
研究生(外文):Chuan-Te Lee
論文名稱:腦垂腺GH3細胞受動作電位波形誘發出L型鈣離子電流之研究
論文名稱(外文):Properties of Action Potential Waveform-Evoked L-type Calcium Currents in Pituitary GH3 Cells
指導教授:吳勝男
指導教授(外文):Sheng-Nan Wu
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:42
中文關鍵詞:L 型鈣離子流動作電位泌乳激素腦垂腺鈣離子
外文關鍵詞:ACTION POTENTIALGH3PATCH CLAMPTRH
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GH3 細胞是A.H. Tashjian Jr.等人於1965年由老鼠腦垂腺前葉腫瘤衍生出來的一種神經內分泌細胞,它可以自發性地分泌泌乳激素 (prolactin),其機制可能與鈣離子依賴性動作電位(Ca2+-dependent action potentials)有關。本實驗的目的是利用whole-cell patch-clamp 方法,檢測GH3細胞在不同波形的動作電位作用之下所產生的L型鈣離子流(L-type Ca2+ current or ICa,L) 的反應。實驗所得結果分述如下:
1.動作電位誘發出來的ICa,L包含兩個部份,第一部份的波峰在動作電位上升段出現,第二部份的波峰在動作電位的下降段出現。
2.延長動作電位下降段的時間,發現經由ICa,L進入的鈣離子流量增加,但其第二部份波峰的高度下降。
3.動作電位停留於波峰的時間增長, ICa,L第二部份的波峰隨之增高。
4.以一連串的動作電位刺激時,導致 ICa,L有不活化的現象。以Ba2+取代溶液中的帶電電荷時,ICa,L的不活化現象明顯減少。
5.以不規則的爆發性動作電位激發出來的ICa,L有不活化的現象。
6.以一連串達到去極化電位的動作電位持續刺激細胞時,鈣離子不只在動作電位發生的過程中進入細胞,在兩個連續動作電位的間隔時間裡,鈣離子也能進入細胞。
7.以方形波刺激時,加入thyrotropin releasing hormone (TRH; 10 µM)所測得的ICa,L比加入前所測得的值大,且其電流與電壓的關係圖略向負方移動。
8.當細胞浸浴於TRH (10 µM),thapsigargin (10 µM),或是cyclopiazonic acid (30 µM),以動作電位誘發出ICa,L時,第二波峰的強度會增強。
9.連串地動作電位刺激使ICa,L不活化的現象,在加入TRH後大為減少。
10.細胞內儲存的鈣離子是否釋放出來,是影響動作電位誘發的ICa,L強度的因素之一。
以上結果顯示,在腦垂腺GH3細胞上以動作電位波形誘發出的ICa,L,其time course以及動力學的表現,與我們以方形波刺激所得到的數據完全不同。改變GH3細胞動作電位的波形以及其激發的形態,可以調節經由L型鈣離子通道進入的鈣離子流量。
The purpose of this study was to examine the time course and kinetics of L-type Ca2+ current (ICa,L) from pituitary GH3 cells in response to various action potential (AP) waveforms using the whole-cell configuration of the patch-clamp technique. The major findings in this study are:
1.ICa,L evoked during the AP waveform exhibited an early and a late component. The early component of ICa,L occurred on the rising phase of the AP, whereas the late component coincided with the falling phase.
2.A prolongation of the falling phase of APs led to an increase in Ca2+ charge carried by ICa,L, although the amplitude of the late ICa,L was reduced.
3.When the peak voltage of AP waveforms was prolonged without changing the rising and falling phases, the amplitude of the late components was significantly increased.
4.ICa,L was also found to inactivate during a train of AP waveforms. When Ba2+ ions were used as the charge carriers, current inactivation during a train of APs was decreased.
5.The amplitude of ICa,L evoked by the AP templates with irregular bursting pattern was inactivated.
6.When spontaneous APs with the depolarizing potentials were replayed to GH3 cells, Ca2+ entry was not only spread over the entire AP, but also occurred during the interspike voltage trajectory.
7.When cells were exposed to thyrotropin releasing hormone (TRH; 10 □M), the ICa,L in response to rectangular pulses was increased and the current-voltage relation was slightly shifted to the left.
8.The presence of TRH (10 □M), thapsigargin (10 □M) or cyclopiazonic acid (30 □M) enhanced the late component of the AP-evoked ICa,L.
9.TRH caused a relief of the inactivation of ICa,L during a train of APs.
10.Ca2+ release from internal stores might also affect the magnitude of AP-evoked ICa,L in these cells.
The results provide evidence that in pituitary GH3 cells, the time course and kinetics of ICa,L during the AP waveforms are distinct from that evoked by rectangular voltage clamp. Changes in the shape and firing pattern of APs in GH3 cells will modulate Ca2+ influx through L-type Ca2+ channels, thus influencing hormonal secretion.
前言......................1
關於patch clamp................1
關於GH3 細胞..................2
細胞對鈣離子的調控...............4
實驗目的....................5
研究方法與材料.................6
細胞培養....................6
電生理.....................6
資料記錄分析..................7
檢驗GH3 細胞L-type Ca2+ current (ICa,L) 的方法.7
動作電位波形的建立...............8
藥物與溶液製備.................9
結果.....................11
方形波與動作電位波形所誘發出來的ICa,L之比較.11
改變動作電位的波形對於ICa,L的影響......12
連續施以動作電位造成鈣離子通道的不活化....13
以達到去極化電壓的自發性動作電位誘發ICa,L..14
比較TRH對方形波與動作電位波形所引發的ICa,L的影響.15
延長動作電位下降段時間所激發出來的ICa,L對TRH的反應. .16
連串的動作電位波形所造成的不活化現象因TRH 而減少..16
細胞內鈣離子儲存量對動作電位誘發的ICa,L之影響..17
討論.....................18
參考文獻...................25
附圖.....................31
圖1.方型波所誘發出的ICa,L,其電流與電壓之相互關係.31
圖2.GH3 細胞受單一動作電位所誘發出的ICa,L....32
圖3.動作電位下降段時間延長對於ICa,L波形的影響....33
圖4.持續時間不同的波峰電壓對於動作電位誘發之ICa,L的影響..34
圖5.以一連串的動作電位刺激細胞所造成的ICa,L的不活化現象..35
圖6.自發性規則與不規則爆發性動作電位對ICa,L的影響....36
圖7.模擬細胞受TRH刺激後產生自發性連續動作電位對ICa,L的影響.37
圖8.TRH 對於方形波或是動作電位波形所誘發出的ICa,L的影響...38
圖9.TRH對延長動作電位的下降段之後誘發出的ICa,L的影響....39
圖10.連續施以動作電位所造成的ICa,L不活化現象受TRH影響而減少40
圖11.TRH , thapsigargin, cyclopiazonic acid 和 dantrolene 對於動作電位誘發出ICa,L的第二部份鈣離子總流量的影響之比較...41
表1.下降段持續時間長度不同的動作電位波形對於GH3 細胞ICa,L的第二部份之影響...........42
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