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研究生:貝瑞發
研究生(外文):Jui-fa Pei
論文名稱:澤瀉萃取物對青蛙皮膜組織短路電流及通透性的影響
論文名稱(外文):Effects of Alismatis Rhizoma''s Extract on Short – Circuit Current and Conductance across Frog Skin Epithelium
指導教授:蕭正夫
指導教授(外文):Chen-fu Shaw
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:2004
畢業學年度:92
語文別:中文
論文頁數:49
中文關鍵詞:澤瀉
外文關鍵詞:Alismatis rhizomavoltage-clampshort-circuit current
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在中藥運用上,澤瀉 ( Alismatis rhizoma ) 屬利水滲濕藥,主要用途為利尿、治療水腫和抗發炎,其作用機轉未見諸文獻報告。利尿作用通常與泌尿系統上皮細胞對鈉離子的重吸收有關,因此推測澤瀉也許能影響皮膜細胞對鹽類及水分通透的作用,本研究之目的即試圖透過短路電流 ( short – circuit current,Isc ) 之測定,檢測上述假設推論是否成立。由於皮膜兩側鈉離子濃度的變化,會導致短路電流改變。本實驗以電壓箝制技術 ( voltage – clamp technique ),探討澤瀉萃取物 ( extracts of Alismatis rhizoma,ARE ) 作用於青蛙腹部皮膜後,對其短路電流 ( Isc ) 及鈉離子通透性的影響。在穩定狀態時,皮膜電位差為64.81 ± 2.44 mV,Isc為59.82 ± 3.58 μA / cm2 ,電導值 ( G ) 為1.09 ± 0.18 μA / cm2 / mV 。在黏膜側加入ARE會使Isc下降約24%,若作用於漿膜側則Isc約降低10%,但兩者電導值都沒有明顯改變。以ARE作用於黏膜側使Isc降低,至達到穩定狀態後,再加入鈉離子通道阻斷劑amiloride,會使Isc進一步下降。反之,若先以amiloride處理黏膜側,再加入ARE則無法使Isc繼續降低。
以一氧化氮 ( nitric oxide,NO ) 的前驅物L-arginine,作用於漿膜側可使Isc增加17.10 ± 9.30 μA/cm2,而能夠直接釋放NO的S-nitroso-N-acetyl-penicillamine ( SNAP ) 卻無法影響Isc。若漿膜側先以ARE或NO synthase抑制劑NG-L-nitro-arginine methyl ester ( L-NAME ) 處理,則可抑制L-arginine造成的Isc升高現象 。
由以上結果顯示,澤瀉在皮膜兩側均可產生作用。在黏膜側使Isc降低的程度,較作用於漿膜側顯著,而澤瀉在黏膜側的作用機制可能與amiloride – sensitive sodium channel有關。至於澤瀉在漿膜側的部分作用,則可能與抑制NO的生成或NO的訊息傳遞路徑有關,間接影響受NO所調控的離子運輸作用。
In traditional Chinese herb medicine, Alismatis rhizoma has been used in treating edema, inflammation and increasing urine flow. Mechanism of Alismatis rhizoma’s effect on these functions has not been elucidated. Since diuresis has been considered closely related to the reabsorption of sodium ion via the epithelium of tubule and collecting duct in kidney, we suspected that Alismatis rhizoma may influence transportation of salt and water.
The measurement of short circuit current ( Isc ) has been used widely to estimate the ion transportation between mucosal and serosal side of epithelium. In the present experiment, we used the voltage – clamp technique to demonstrate the effect of Alismatis rhizoma’s extracts ( ARE ) on Isc and sodium ion conductance in frog skin. Our result showed that in control stage, the potential difference ( PD ) of frog skin is 64.81 ± 2.44 mV, the Isc is 59.82 ± 3.58 μA / cm2 and the conductance is 1.09 ± 0.18 μA / cm2 / mV.
After ARE was applied to mucosal side of the frog skin, its Isc decrease from 62.63 ± 5.31 μA / cm2 to 47.92 ± 5.41 μA / cm2, which could further go down to 3.36 ± 1.06 μA/cm2 by adding amiloride. Treating serosal side of frog skin with ARE decreased approximately 10% of its Isc. No apparent changes in conductance were observed by adding ARE to mucosal ( 0.98 ± 0.24 μA / cm2 / mV ) or serosal side ( 0.96 ± 0.23 μA / cm2 / mV ).
Adding L-arginine ( the precursor of nitric oxide ) to the serosal side of the skin epithelium elevated the Isc for 17.10 ± 9.30 μA/cm2. This effect can be inhibited by applying ARE or NG-L-nitro-arginine methyl ester ( L-NAME, NO synthase inhibitor ) before application of L-arginine.
In summary, Alismatis rhizoma could affect Isc on both mucosal and serosal sides of the frog skin. Its effect on lowering Isc was more obvious when applied to the mucosal side than to the serosal side. The ARE may exert its effect on mucosal side by affecting amiloride–sensitive sodium channel and on serosal side by affecting the NO signal transduction pathway.
目 錄
頁次
目錄-----------------------------------------I
圖表目錄------------------------------------Ⅱ
中文摘要-------------------------------------1
英文摘要-------------------------------------2
文獻回顧及研究目標---------------------------4
實驗材料及方法------------------------------12
結果----------------------------------------18
討論----------------------------------------21
結論----------------------------------------27
參考文獻------------------------------------28

圖表目錄
頁次
表一. 本實驗各種藥物對皮膜作用後的短路電流 ( Isc )、電
導值 ( G ) 以及短路電流變化百分比( DIsc )列表--33
圖一. 澤瀉部分組成份之化學結構--------------------34
圖二. KJU 模型及電壓箝制 ( voltage – clamp ) 示意圖---35
圖三. 澤瀉作用於黏膜側及漿膜側對短路電流的影響----36
圖四. Amiloride 與澤瀉的配對作用-------------------37
圖五. L-Arginine 作用在漿膜側以及黏膜側對短路電流的影
響----------------------------------------38
圖六. 澤瀉與L-NAME對L-arginine 造成Isc 升高的抑制作用
-----------------------------------------39
圖七. SNAP 作用於漿膜側對短路電流的影響----------40
圖八. 漿膜側的鹼性環境對短路電流的影響------------40
圖九. 澤瀉及Amiloride 在黏膜側個別作用和配對作用時,對
短路電流影響之統計圖------------------------41
圖十. 澤瀉作用於漿膜側對於短路電流影響統計圖------42
圖十一.L-Arginine、SNAP 以及KOH 作用於漿膜側對短路
電流影響統計圖---------------------------43
圖十二.L-NAME 以及澤瀉在漿膜側抑制L-arginine 作用之統
計圖---------------------------------------44
圖十三.體積濃度2%之酒精溶液對皮膜短路電流的影響--45
圖十四.酒精濃度序列增加對黏膜側短路電流的影響-----46
圖十五.黏膜側的酸性環境對短路電流的影響-----------46
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