在本論文中，為了了解吸附在細胞膜上的蜂毒胜肽形成孔洞的機制，除了已 商業化的共聚焦螢光影像偵測方式與漸趨成熟的螢光相關光譜技術，我們更建立 一套系統與測量方法提供即時觀測單一脂質體受到蜂毒胜肽攻擊後的單點螢光 變化，且設計簡單，無樣品製備繁複的困擾。
我們利用自行架設的共聚焦螢光顯微系統，測量不同濃度下的脂質體內部螢 光變化情形，並將結果中的突峰進行參數與統計分析，我們推測蜂毒胜肽有單分 子的攻擊行為。從共聚焦顯微鏡得到幾種不同的螢光變化結果：螢光忽然增強又 漸漸變弱、螢光慢慢增加直到內外相同。我們可以知道：細胞膜與蜂毒胜肽接觸 後會增加細胞膜的通透性。另外，我們也提出三種峰毒胜肽攻擊行為的假設。
我們也利用螢光相關光譜技術量測細胞膜的流動性變化，發現與蜂毒胜肽接 觸後，細胞膜的流動性有顯著的變慢現象，且此影響有一定的限度，並非與濃度 成正比，這可能代表蜂毒胜肽在細胞膜上的分布密度已達飽和，增加蜂毒胜肽，
也無法再繼續影響細胞膜流動性。
針對本論文的實驗設計，我們可以做一些修改來增進測量的穩定度與提高訊 雜比。混合樣品時，我們可以在玻片表面與脂質體上修飾官能基，將脂質體固定 位置，降低漂動造成的螢光擾動。我們也可以對脂質體進行粒徑的篩選，鎖定觀 測單一粒徑的脂質體有助於做定量的分析。
在本論文中，我們使用多種螢光技術觀察單一脂質體與蜂毒胜肽作用的即時 螢光影像、強度變化、與膜流動性變化，未來我們也可以利用這些技術觀察不同 細胞膜成分的脂質體或是不同胜肽的攻擊行為作為本實驗的延伸。
附錄
附錄一 實驗相關藥品
附錄表一 實驗藥品
藥品 說明 製造商
POPC 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine 16:0/18:1(9Z) PC
Avanti, U.S.A.
CHCl3 Chloroform J.T. Baker,
U.S.A.
DiD Lipophilic dye,
Sub-cellular localization: cell membranes and lipids, Abs/Emi: 648 nm/670 nm
Molecular Probes, U.S.A.
MeOH Methanol J.T. Baker,
U.S.A.
Glucose D-(+)-glucose,
≥99.5% (GC)
Sigma, U.S.A.
NaCl Sodium chloride
≥99.5% (titration)
Sigma, U.S.A.
CF 5(6)-carboxyfluorescein, Abs/Emi: 492 nm/517 nm
Fluka, U.S.A.
R6G Rhodmine 6G chloride Abs/Emi:428 nm/551 nm
Molecular Probes, U.S.A.
Alexa633 Abs/Emi: 632 nm/647 nm Molecular
Probes, U.S.A.
Melittin The main component of the honeybee venom, Mr: 2846.50,
Purity: > 97% by reverse phase HPLC
Bachem, Switzerland
58
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