建立並評估生物技術平台篩選具抗發炎效果食品

Abstract

慢性發炎為一種長期且低度的炎症反應，發炎物質長時間釋放的結果會造成人體各部位細胞之損傷，因此導致許多慢性疾病如粥狀動脈硬化、第二型糖尿病和癌症。近期研究指出核轉錄因子Kappa B(NF-κB)的活化可促進發炎細胞激素如腫瘤壞死因子(TNF-α)、介白素(Interleukin-6, IL-6)的生成。此外，也增加發炎反應酵素如誘導型一氧化氮合成酵素(Inducible nitric oxide synthase, iNOS)、環氧化脢二號 (Cyclooxygenase-2, COX-2)的合成，因此NF-κB為慢性發炎的重要啟動分子。目前，具抗發炎功效之食品成份的開發已成重要議題。一般而言，Western blot 和RT-PCR是最常被用以檢測與發炎相關蛋白質與RNA變化的方法；但其成本、時間與技術考量明顯較繁雜。本報告利用螢光酵素(Luciferase) 報導基因為基礎，建立可以快速篩選抗發炎食品的細胞平台。在這平台當中，利用慢性發炎反應中NF-κB的活化及其下游基因iNOS和COX-2被表現的分子特徵，我們構築了可反映上述三個基因表現的質體，利用Luciferase作為報導基因，並進一步將此質體放入RAW264.7巨噬細胞中，利用質體抗Hygromycin特性進行篩選，並在其中挑選出不同的反應株進行測試，成功挑選了在發炎反應下可表現報導質體而產生冷光的細胞殖株，並利用發炎反應的抑制劑檢測本平台的效應，結果證明本平台的結果與傳統方法的結果相符。此外，食物萃出之已知抗發炎物質川陳皮素亦證明可降低本平台的發炎指標。而利用本平台作不同溶劑萃取的橄欖萃出物篩選的結果顯示，橄欖之正己烷粗萃物在脂多醣誘導發炎的平台中抑制效果最佳。因此確定本平台未來可用於進行高通量之篩選，以期可以更快速篩檢具抗發炎功效之食材。

Chronic inflammation leads to a progressive inflammation in certain types of cells. And it is responsible for disorders such as heart disease, diabetes and cancer. Moreover, recent studies report that the activation of nuclear factor kappa B (NF-κB) increases the expression of inflammation-related protein such as tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), which further enhance the chronic inflammation, thus conduct the development of disorders. Therefore, food with anti-inflammation activity is commonly used to improve chronic inflammation. Traditional analyses for estimating the anti-inflammation activity of food components are western blot and RT-PCR, which are used to detect the expression of inflammation and proimflammation genes. The whole process is both time and money consuming. In order to develop a better method, we fused the promoter regions of iNOS and COX-2 were to the upstream of luciferase gene, respectively, thus the expression of luciferase represents the expression of iNOS and COX-2 accordingly. Similarly, transcriptional activity of NF-κB can be estimated by using the reporter construct containing the response element of NF-κB. Using hygromycin selection, we have successfully obtained the reporter plasmid expressing clones, which can be induced by LPS, an inflammation inducer. We further estimated the effect of these cell platforms by treating anti-inflammation reagent. The results reveal that our platforms can respond to both inflammation and anti-inflammation stimulation, and the response of our platform is similar to the result of real time PCR. In addition, the known anti-inflammation factor from food, such as nobletein has been proven to decrease the inflammatory effect of LPS on our platform. We further used the platform to screen components with anti-inflammation ability. The screening result of olive extract showed that the olive hexane extract exhibited better anti-inflammation activity than the other solvent extracts. Therefore, we conclude that the platform is effective in large scale screening.