以標的螢光探針研究巨噬細胞內之過氧化氫

Abstract

活性氧化物(reactive oxygen species, ROS)為具有高度氧化力的分子。ROS之生成可由細胞內或細胞外物質所誘發。當細胞內的ROS過量時會攻擊DNA、蛋白質和細胞膜脂質而造成無法修復的傷害，此與癌症、老化及疾病生成有關。在所有的活性氧化物中，過氧化氫 (H2O2) 被認為是最主要的分子，因為過氧化氫除了調節致病的機轉之外，目前已經有研究指出，過氧化氫也是一些訊號傳導路徑中的二級傳訊者。然而在多樣性的生物環境中，研究有關活性氧化物的過程一直遭遇一些無法克服的挑戰；主要的原因是因為在活體系統中，缺少對於活性過氧化物的專一性探針及研究方法。 在本篇研究中，我們首要目的是設計兩種化學螢光物質，我們稱之為探針一與探針二。這兩個探針都可以對過氧化氫有專一性的反應，並釋放出藍色的螢光物。因此我們可以利用此探針，在活體細胞中偵測出細胞面對壓力時所釋放出的過氧化氫。我們希望利用此篇研究的方法，建立一個簡單、快速、直接的平台，來偵測活化的巨噬細胞內之過氧化氫濃度;並藉由螢光儀偵測巨噬細胞內的相對螢光強度來定量。此外在未來的應用上，也可以利用此平台來尋找具有抗氧化劑活性的潛在化合物。

One major contributor to oxidative damage is hydrogen peroxide (H2O2), which has been considered mostly as reactive oxidative species (ROS), and mediate pathogenic processes. In addition, H2O2 is emerging as a newly recognized as a secondary messenger in cellular signal transduction. However, a substantial challenge in elucidating its diverse roles in complex biological environments is the lack of methods for probing this reactive oxygen metabolite in living systems specifically. Here, we reported the synthesis and application of Probe 1 (1a and 1b), two new fluorescent probes that showed high selectivity for H2O2 and were capable of visualizing the addition of exogenous H2O2 to living cells. These probes were able to serve as a tool for evaluating the H2O2 production in complex physiological and pathological processes. The main purpose of this study was to create an easy, time-saving, and a straightforward platform for measurement the exact H2O2 concentration in living RAW 264.7 cell. Through quantitating the relative fluorescence intensity in vivo by spectrofluoromerty, we might discover new small molecules for potential antioxidants in the future.