臺灣博碩士論文加值系統

Adenosine為中國傳統藥方靈芝抗血小板凝集作用的主要成份之一，本實驗設計主要是以Adenosine進行化學結構分子修飾，將改變支鏈後之Adenosine衍生物進一步探討其藥理活性。
化學修飾的部份在以Adenine為主體結構上之第四和第八位置上進行新鍵結，最後合成出新型的Adenosine衍生物：Adeno-1、Adeno-2與Adeno-3，再運用活體與離體動物實驗及人體之血小板，來探討Adeno-1、Adeno-2與Adeno-3的抗血小板作用與血管鬆弛作用機制。
在活體心臟血管實驗的部份：由靜脈分別注射Adeno-1、Adeno-2與Adeno-3各1.0 mg/kg於經pentobarbital麻醉之正常血壓Wistar系大鼠，注射Adeno-1或Adeno-2所引起的血壓與心跳改變並不明顯，而注射Adeno-3後可使血壓些微下降，在心跳的改變部份也是並不明顯。
在離體組織實驗的部份：於phenylephrine誘導收縮的大鼠離體胸主動脈血管，分別累積投與Adeno-1、Adeno-2與Adeno-3 （從10-9 ∼ 10-4 M）後，對於去除內皮細胞或是保留內皮細胞的血管，均可產生劑量相關性的血管鬆弛作用。其中Adeno-3之血管鬆弛作用會因內皮細胞的去除及一氧化氮合成酶(nitric oxide synthase ; NOS) 抑制劑L-NAME (100 µM)的加入而降低其作用，Adeno-3的血管鬆弛作用亦受到soluble guanylate cyclase （sGC）抑制劑ODQ (1 µM) 、鉀離子通道阻斷劑tetraethylammonium chloride (TEA，10 mL)、4-aminopyridine (4-AP，100 µM) 以及 charybodotoxin (ChTX，0.1 µM)等藥物之前處理而抑制，但在glibenclamide ( 1 µM) 之前處理部分則無明顯抑制作用。
在大鼠離體胸主動脈，投與Adeno-3後對於80 mM高鉀溶液所引起之血管收縮之鬆弛作用可明顯遞減。在以 Adeno-3 (10- 6 M) 前處理後，對於不同濃度( 10-9、10-8、10-7 M )的sodium nitroprusside ( SNP) 所引起之血管鬆弛，Adeno-3亦有增強之作用。
在抗血小板凝集之作用方面，我們比較了Adeno-1、Adeno-2、Adeno-3 及YC-1的強度差異，發現在200 µM的濃度下，四者均能有不同程度的抑制膠原蛋白（collagen，10 µg/ml），sodium arachidonate（100 µM），adenosine diphosphate（ADP，20 µM），血小板活化因子（platelet— activating factor，PAF，2 ng/ml），serotonin (5-HT，4.2 µM)、凝血酵素（thrombin，0.1 U/ml）、epinephrine (5 µM) 等所誘發的人體血小板凝集作用。
其次，以放射性免疫分析方法（RIA）測定來評估cGMP與cAMP之釋放量，利用［125I］cGMP與［125I］cAMP進行分析，(1) 在人體血小板的部分：Adeno-1、Adeno-2、Adeno-3 及YC-1四者（濃度分別從 1 ∼ 100 µM），對於血小板內cGMP與cAMP的釋放量，呈現出劑量相關性的增加含量。(2) 在大鼠離體胸主動脈平滑肌細胞的部分：可發現Adeno-3 呈現劑量相關性增加細胞內cGMP 的釋放量，而在分別投與L-NAME (100 µM), Methylene Blue (100 µM)及ODQ (10 µM)等前處理藥物後，則cGMP 的釋放量明顯減少。
在磷酸二酯酶的活性分析測定方面 ，從人類血小板之細胞中可分離出PDE5的isozyme，由測試的數據指出Adeno-1、Adeno-2與Adeno-3 可分別抑制PDE5約 50%、13%、 71% 相對於IBMX活性的抑制濃度。
基於以上活體、離體組織實驗，以及進一步進行放射性免疫分析方法，可以顯示Adeno-1、Adeno-2及Adeno-3之抗血小板凝集作用可能是抑制PDE5，增加cGMP的釋放而產生作用。而在血管平滑肌之鬆弛作用方面，Adeno-3可能是部份經由刺激一氧化氮的產生，活化sGC ，開啟鉀離子通道而增加cGMP 釋放，但是大部分的作用很可能是來自於抑制PDE5所產生的影響。

Numerous studies have shown that the Ganoderma lucidum can improve human immune-system, anti-hypertension, prevent blood coagulation, and anticancer effect, etc. Adenosine is one of the main compounds of the Ganoderma lucidum to have the anti-platelet aggregation activities.
Adenosine derivatives, Adeno-1, Adeno-2, and Adeno-3 were designed and competited with YC-1 about their anti-platelet aggregation activities and vasorelaxant effects. Evidences indicated that Adeno-1, Adeno-2, and Adeno-3 (from 0.2 ~ 200 M) had concentration-dependently inhibited arachidonic acid (AA)-, collagen-, epinephrine-, ADP-, thrombin-, serotonin (5-HT)-, and PAF-induced platelet aggregation.
In phenylephrine- preconstricted endothelium-intact or denuded rat aortic rings, Adeno-3 produced concentration-dependent relaxations. The relaxation was reduced by endothelium removal or by the presence of L-NAME (100 μM), a nitric oxide synthase inhibitor. In addition, the vasorelaxant effect of Adeno-3 was inhibited by pretreatment with a soluble guanylate cyclase inhibitors ODQ (1 μM), a K+ channels blocker TEA (10 mM), a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 μM), and charybdotoxin (ChTX, 0.1 μM), while not significantly different with a KATP channels blocker glibenclamide (1 μM). Moreover, increased extracellular potassium levels (80 mM high potassium solution) resulted in an attenuation of the concentration-dependent vasodilator effects of Adeno-3. Preincubation with Adeno-3 could enhance the vasodilator response to the exogenous NO-donor SNP.
In isolated rat atria, Adeno-3 didn’t produce inotropic or chronotropic activities. While the depressive effect was revealed at concentration above 10-5 M.
Adeno-3 (with 0.1, 1, 10, 100 μM) induced concentration-dependently increases in intracellular cyclic GMP levels in rat aortic smooth muscle cells (RASMC). When pretreated with L-NAME (100 μM), methylene blue (100 μM) or ODQ (10 μM) before Adeno-3 (100 μM) , the contents of cyclic GMP was decreased.
In phosphodiesterase inhibitor assay, Adeno-1, Adeno-2, and Adeno-3 were inhibited about 50%, 13%, and 71% activities related to the IBMX inhibited activities.
It is suggested that the smooth muscle relaxant effects of Adeno-3 might be mediated by the stimulation of NO / sGC / cGMP pathway, the opening activity of the K+ channel and PDE5 inhibited effents. In human platelet, the Adeno-3 is able to inhibit the platelet aggregation activities might be mediated by the PDE5 inhibited effents.

1. 摘要 ---------------------------------------------- 1
2. 英文摘要 ------------------------------------------ 4
3. 縮寫表 -------------------------------------------- 6
4. 緒論 ---------------------------------------------- 7
5. 研究材料 ----------------------------------------- 10
6. 研究方法 ----------------------------------------- 14
7. 研究結果 ----------------------------------------- 23
8. 討論 --------------------------------------------- 29
9. 參考文獻 ----------------------------------------- 34
10.附圖與附表---------------------------------------- 39

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