第四章 實驗部份
第四節 藥理活性測定方法
A、抗血小板凝集活性測定 血小板凝集引發劑之製備：
1 Thrombin (bovine)：
購自 Park Davis Co.USA,溶解於 50%（v/v）glycerol 製備成 100 NIH units / mL 之 stock solution。
2 Arachidonic acid（AA）：
購自 Sigma Chemical Co. USA，以去離子水溶解之.
3 Collagen （type 1, bovine Achilles tendon）：
購自 Sigma Chemical Co.USA， 溶解在 25 ml acetic acid 中， 於 4 ℃ 研磨使均質化， 以 1 mg / mL 的濃度儲存於 –70 ℃。
4 Platelet-activating factor（PAF）：購自 Sigma chemical Co.USA，
溶於 chloroform 中， 儲存於-20 ℃使用前以 0.9 ﹪NaCl 稀釋之。
5 Adenosine diphosphate (ADP)：購自 Sigma chemical Co.USA，以 去離子水溶解備用.
血小板懸浮液（platelet suspension）之製備：
從兔耳靜脈抽出血液， 與 100 mM EDTA 以 1：14（v/v）的比例混 合，混合後， 室溫下立即以 90 ×g 離心 10 分鐘， 取出上層富含血小板 之血漿， 再以 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（BSA, 3.5 mg / mL）的 Tyrode 溶液懸浮之， 並於相同轉速下離心 10 分鐘。所得血小板在不含 EDTA 的 Tyrode 溶液懸浮後，再於相同條件下離心後， 取血小板層， 將其懸 浮於 Tyrode 溶液中， 並以 Coulter counter（Model ZM）計數，調整血小 板數目約為 4.5 × 108 platelets / ml， 最後加入 1 mM 鈣離子， 靜置 30 分鐘後， 再進行實驗。Tyrode 溶液除了含 BSA 外, 其他組成如下（mM）： NaCl （136.9）， KCl（2.7），MgCl2 （2.1），NaH2 PO4 （0.4）， NaHCO3
（11.9）及 glucose （11.1）。
血小板凝集試驗:
採用 Lumi-aggregometer（Model 1020, PayLon， Canada）及混濁度 法（turbidimetric method）之原理來測定凝集程度59。 將血小板懸浮液 0.4 毫升加入經 silicone 包衣的小玻璃管中，再加入適量的 Tyrode 溶液， 在 37℃下以小磁棒做每分鐘 1200 轉的攪拌， 加入凝集引發劑後， 觀察 6 分鐘， 若未特別說明， 均在加入凝集引發劑前三分鐘加入測試化合物。
凝集程度的計算是以 PBS 的吸光度作為 0﹪的血小板凝集， 以 Tyrode
溶液的吸光度作為 100﹪的血小板凝集，利用下列公式計算之60： 凝集 ﹪＝【（A1-A2）/（A1-Ab）】×100﹪
A1：加入凝集引發劑前的吸光度 A2：加入凝集引發劑後的吸光度 Ab：Tyrode，s solution 的吸光度
B、抗炎活性試驗測定方法
中性白血球（neutrophils）之製備：
大鼠（Sprague Dawley，300-350 g）經 pentobarbital（60 mg / kg, i.p.）
麻醉後，由腹腔動脈抽血，與 dextran 混合靜置， 經 Ficoll-hypaque density gradient 離心沉降後， 將 neutrophils 和其它血液細胞分離.並在低張溶液
（0.05 ﹪saline）下除去紅血球， 將細胞於包含 0.25 ﹪bovine serum albumin 之 1.75 ﹪saline 清洗後，並懸浮於 Hanks，balanced salt solution
（HBSS）， 組成為 4 mM NaHCO3及 10 mm N-(2-hydroxyethyl) piperazine-
N
，-(2-ethane sulfonic acid)（HEPES） (pH7.4)成每毫升 2 ×10 6個細胞，其中包含 90-95﹪之 neutrophils。
測定方法：
Neutrophil degranulation method
（1） β-Glucuronidase 釋放之測定61：
將中性白血球懸浮液分別與 DMSO 或檢品溶液於 37℃培養三分鐘 後， 各別加入 fMLP（10 mM）與之作用， 經四十五分鐘後， 加入冰 浴過之 Tyrode’s solution 來中止反應， 混合液經離心（1000 xg）十分鐘 後， 將上清液取出， 利用上清液中之β-Glucuronidase 與 phenophthalein-γ-D-glucuronide 反應後， 在分光光度計 550 nm 波長下來測量所含β -Glucuronidase 之量。
（2） Lysozyme 釋放之測定62：
同（1）中處理後，將所得含 Lysozyme 之上清液， 利用
Micrococcuslysodeikticus 作為受質， 經由分光光度計在 450 nm 處測量之。
β-Glucuronidase 及 lysozyme 的釋放表示如下：
﹪release＝【（release elicited by secretagogue – spontaneous release）/total
content】×100
Total content 是將細胞懸浮於 Triton X-100 後所測量之值。
C、Superoxide formation method Superoxide anion 釋放之測定：
於檢品及參考溶液（0.4 ml）中含有 0.2 ml 細胞懸浮液（5 ×106 cells mL）及 0.9 mg / ml 的 ferricytochrome C， 而參考溶液中再加入 12.5 mg / ml 的 superoxide dimutase（SOD）. 將其於 37 ℃培養三分鐘後， 分別加 入 fMLP（0.3μM）與之作用， 經 30 分鐘後，離心取上清液，利用分光 光度計在 550 nm 測定 superoxide dismutase， 從可抑制之吸光部分, 即可 知中性白血球釋放過氧化物游離根之程度63。
O2-（superoxide anion）之量是以下式計算之：
O2
-（nmol）＝19.08 × absorbance
D. 抗過敏活性試驗方法 肥大細胞（mast cells）之製備：
大鼠經頸部放血後（exsanguinated rats; Sprague-Dawley, 250-300 g），
取 10 ml 含肝素之 Tyrode’s solution（O2
- Tyrode’s solution A）注入鼠之腹 腔內， 按摩 1-2 分鐘並取出腹腔溶液，經 38 ﹪牛血清蛋白之 glucose- free Tyrode solution 處理離心後， 沉澱細胞經清洗後。 並懸浮於 Tyrode’s solution B，其組成如下（mM）：NaCl（137），KCl（2.7），NaHCO3（12），
MgCl2（1.0），NaH2PO4（0.3），CaCl2（1.0），glucose（5.6）and bovine serum albumin 0.1 ﹪. 巨大細胞數目調整成每毫升 1-1.5 ×106個細胞，並測定 細胞存活率.
Mast cell degranulatiopn method
（1） 組織胺（histamine）釋放之測定64：
將肥大細胞懸浮液分別與 DMSO 或檢品溶液於 37 ℃培養三分鐘 後，個別加入 compound 48 / 80（10mg / ml）與之作用， 經十五分鐘後，
加入冰浴過之 Tyrode’s solution 來中止反應， 混和液經離心（1000 xg）
後將上清液取出， 利用上清液中之 histamine 與
o-phthaldehyde 反應後，
以螢光分光光度計在 350 / 450 nm 處來測量所含組織胺之量。
（2） β-glucuronidase 釋放之測定62：
同（1）中處理後, 將所得含β-glucuronidase 之上清液， 利用 phenolphthalein-β-D-glucuronide 作為受質， 經由分光光度計在 550 nm 處測量。
Histamine 及β-glucuronidase 的釋放表示如下：
﹪relrase＝【（release elicited by secretagogue-spontaneous release）/ total content】×100
Total content 是將細胞懸浮於 Triton X-100 後所測量之值。
E. NO (nitrtic oxide)及 TNF-α活性測定 1. 材料
(1) Iscover's Modified Dulbecos Medium 與牛胚胎血清(fetal bovine serum，FBS)：購自 Gibco BRL (Gaithersburg)
(2) TNF-β酵素免疫分析(enzyme immunoassay, EIA) kit：購自 Genzyme Co. (MA)
(3) 老鼠干擾素-α(mouse interferon-α, INF-α):購自 R＆D System (MN)
(4) RAW 264.7 mouse macrophage-like cell line ：購自 American Type Culture Collection, MD.
(5) Dubecco's Modifed Eagle Medium (DMEM)與犢胚胎血清(fetal calf serum)：購自 Gibco BRL (Gaithersburg)
(6) 其餘試藥皆購自 Sigma (St. Louis, MO) 細胞培養與前處理51
Murine microglia cell lines N9 係以含有 2 ﹪FBS 與抗生素之 Iscover's Modified Dulbeco's Medium 培養，在前處理部份，先將待測藥物與細胞於 37 ℃混合一小時，再以 100 ng / ml 的 LPS (Escherichia coli, serotype
0111：B4) 加 100 U/ ml INF-α刺激 24 小時，最後保存-70 ℃需要時才取 出。
RAW 264.7 mouse macrophage-like cell 則以含有 5 ﹪fetal calf serum、100 units / ml penicillin 與 100 ìg streptomycin 之 DMEM 培養，在前處理部 份，先將待測藥物與細胞於 37 ℃混合一小時，再以 100 / ml 的 LPS
(Escherichia coli, serotype 0111：B4) 加 100 U / ml INF-γ ，刺激 24 小時，
最後保存-70 ℃需要時才取出。
NO 之測定 65
於 150μl 之培養基中，依序加入 sulfanilamide ( 5 mM, 40μl )、HCl (2 M, 10 μl) 及 naphthylethylenediamine (40 mM, 20μl)，室溫下混合 10 分鐘 後，以 microplate 計數器在 550 nm 下測其吸光度。一氧化氮標準曲線，
則以 NaNO2 於相同條件下所求得。
TNF-α之測定
培養基中的 TNFα-之測定使用 TNF-α enzyme immunoassay (EIA) kit，
依照廠商所提供之操作步驟進行測定。
F. 抑制癌細胞之活性
HL 60 人類血癌細胞之培養66-67
將 HL 60 人類血癌細胞置於 RPMI-1640 medium (含 10 % FBS，100 U/ml penicillin，100μg / ml streptomycin，1 % L-glutamine)，在 37 ℃ ， 5 % CO2，溼度 95 %之 incubator 中培養；細胞密度控制在 2-5 ×105 cells /ml，每次加入新鮮 medium 的同時，以 trypan blue exclusion 方式確定 細胞的存活。活細胞不會讓 trypan blue 進入細胞中，而死亡細胞則會因 trypan blue 的進入呈現淡藍色，經由血球計數盤 (hemocytometer)計算 得知細胞數。
Carbazole 類化合物對培養中 HL60 人類血癌生長速率影響之測試 把 HL-60 人類血癌細胞培養於 24well plate 中(細胞密度為 2.5 ×105 cells/ml)，分別加入不同濃度之 Carbazole 類衍生物，置於 37 ℃ ，5 % CO2，溼度 95 % 之 incubator 中培養 24 小時，取出 plate，經由離心除 去上清液，以適量 PBS (phosphate-buffered saline) 清洗細胞，離心 再除去上清液，加入 4μg /mL propidium iodine (PI)，利用流式細胞 儀 (flow cytometry)分析之。
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發表投稿之論文
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