藥理試驗方法

一. 抗血小板凝集活性試驗

(一)血小板凝集引發劑之製備

(1) Thrombin–購自 Park Davis Co. USA，溶解於 50% (v/v) glycerol 中，製備成 100 NIH units/ml 之 stock solution。

(2) Arachidonic acid (AA)–購自 Sigma Chem. Co. USA，以去離 子水溶解之。

(3) Collagen (Type I bovine Achilles tendon)–購自 Sigma Chem.

Co. USA，4 ℃下於 25 毫升醋酸中研磨均勻，以去離子水稀 釋成 1 mg/ml，儲存於-70 ℃下。

(4) Platelet-activation factor (PAF)–購自 Sigma Chem. Co. USA，

溶於氯仿，儲存於-20 ℃，使用前以 0.9% NaCl 稀釋之。

(5) Adenosine diphosphate (ADP)–以去離子水溶解備用。

(二)血小板懸浮液(Platelet suspension)之製備

將 EDTA 與兔耳靜脈抽出血混合後，使 EDTA 之最終濃度 為 6 mM，在室溫下即以 90 × g 離心 10 分鐘，取出上層富含血 小板之血漿(platelet-rich plasma)，再將其以 500 × g 離心 10 分鐘，

除去血漿後，將下層血小板以含有 EDTA (2 mM)及 Bovine serum albumin (3.5 mg/ml)的 Tyrode's solution (calcium free)清洗之，在

於相同轉速(500 × g)下離心 10 分鐘，所得之血小板以不含有 EDTA 之 Tyrode's solution 清洗之，再於相同之條件下離心後，取 血小板層，將其懸浮於 Tyrode's solution，其組成如下(mM)：NaCl

(136.8)、KCl (2.8)、NaHCO₃ (11.9)、MgCl₂ (1.1)、NaH₂PO₄ (0.33)、

CaCl₂ (1.0) and glucose (11.2)，並以 Coulter counter (Model ZM)計 數，調整血小板數約為 4.5 × 10⁸ platelet/ml 左右，最後加 1 mM 鈣離子(Ca⁺²)放置 30 分鐘後，進行實驗。

(三)血小板凝集(platelet aggregation)之試驗

利用混濁度法(turbidimetric method)之原理來測定凝集程度 ³⁹，並以 Lumi-aggregometer (Model 1020, PayLon, Canada)測定 之。將血小板懸浮液 0.4 毫升加至經 silicone 包衣的小玻璃管之 中，並以小磁棒做每分鐘 900 轉(900 rpm)的攪拌，若未特別說明，

均在加入樣品三分鐘後，再加入凝集引發劑，六分鐘後觀察結果。

為了排除溶媒(DMSO)影響，在血小板溶液的濃度為 0.5%，全部 反應過程皆在 37 ℃下進行，凝集程度的表示方法如下 ⁴⁰：

凝集(%)=[(A1-A₂) ÷(A₁-A_b)] × 100%

A₁ =加引發劑前的吸光度 A₂ =加引發劑後的吸光度 A_b =Tyrode’s solution 的吸光度

二. 裸鼠的血管新生實驗

從國家實驗動物中心買進裸鼠，其每隻重約 20 克，並依所需之 情況不同，而將裸鼠分成數組，將其飼養於特殊實驗室(無菌實驗室) 數日，才開始進行以下實驗。裸鼠的血管新生實驗，乃是將 matrigel 在 4 ℃液態狀時，混合 vehicle 或是 thrombin，若需觀察藥物的作用，

則將所需之藥物劑量混合入 matrigel 中，以皮下注射方式打入裸鼠

左下腹部(0.5 ml)，經過六天後將裸鼠犧牲，小心的將 matrigel 部位 剪下，拍照存證，若是 vehicle 組，則 matrigel 並無任何血管新生發 生，若是 VEGF 組，則 matrigel 處有明顯之血管新生發生(Figure 5)。

此外，並將此 matrigel 部位減成相等之兩半，一半做病理組織切片 (H&E 染色)，一半做 hemoglobin 含量測定，來將血管新生作用量化。

Basal Control YD-3 (30 µM) (Thrombin 2 U/ml) + thrombin (2 U/ml)

Figure 5

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