造成組織細胞壞死並引發嚴重的發炎反應是蛇毒心臟毒蛋白眾多的病理反應之一，然而，文獻上卻欠缺有關造成此一反應的作用模式及其分子機制之相關研究。在本篇論文中，我們利用人類嗜中性白血球以及老鼠心肌細胞株兩種細胞的生理特性，建立一個實驗模式，以直接和間接的方法嘗試了解蛇毒心臟毒蛋白對細胞的作用機轉及其誘發發炎反應的可能途徑，並且透過自製心臟毒蛋白親和管柱的專一性，來尋找細胞膜上可能與心臟毒蛋白發生作用的結合蛋白。我們初步的實驗結果顯示，於濃度梯度下並不會對人類嗜中性白血球造成明顯的化學趨化功能，但可透過自體分泌及/或旁分泌的間接方式趨化並活化自身或其他免疫細胞。藉由親和管柱的實驗，我們在嗜中性白血球細胞膜上找到四個心臟毒蛋白可能的目標蛋白，分別為乳鐵蛋白、細胞自溶酵素G、彈性蛋白酶2及分子量約60 kDa的未定義蛋白，此外，我們利用蛋白酶原法，發現心臟毒素會引起嗜中性白血球釋放出基質金屬蛋白酶9及絲胺酸蛋白酶。上述的結果，使心臟毒蛋白對細胞的作用機制及發炎反應的研究推進了一步，由我們建立的實驗模式，也有助於研究嗜中性白血球因應發炎反應的機制。

Cardiotoxins (CTXs), most constituents of cobra venoms, was found to cause inflammatory action. In the present study, we found that toxin γ can rapidly bind to human polymorphonuclear neutrophils (PMNs) and subsequent induce inflammatory response and its possible binding targets were isolated and identified from cell surface-bound fractions. Toxin γ itself did not activate significant PMNs oxidative and chemotactic ability comparing to a well-established chemoattractant fMLP under the cytotoxic dosage. However, the PMNs were dramatically attracted by a modified Boyum assay in which the target cell, such as human PMNs, differentiated macrophages and mouse cardiomyocyte cell line were first treated with toxin respectively and the releasing chemicals were used as the assaying attractants, indicating that CTX exert its inflammatory ability, if not all, via a autocrine and/or paracrine mechanism. When PMNs were exposed to toxin γ, the binding constant was estimated to be 1 μM by a direct binding assay using FITC-conjugated toxin γ and the time course of such binding revealed a rapid and saturated interaction with a T1/2 in 5 minutes. We isolated four putative Tγ-binding proteins from PMNs cell surface-bound fractions and identified them respectively to be lactoferrin, Cathepsin G, elastase 2 and one novel protein without certain identity with a MW near 60 kDa, by the methods of MS/MS peptide search and/or protein N-terminal sequencing. Interestingly, all these proteins are serine proteinases and the caseinolytic activity of the serine proteinases and metalloproteinases can also be detected in the cell-free supernatant, whereas the corresponding control has no effect. These findings provide an insight into the inflammatory properties of CTX and suggest a novel mechanism for illustrating neutrophils action in response to CTX.

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