以NF-IL6及NF-κB為導向之藥物篩檢系統的建構與其在中草藥研究上的應用

Abstract

在藥物的篩選模式中，轉錄因子 ( transcription factor ) 及其可調控的啟動子區域 ( enhancer or promoter ) 皆是常被拿來作為篩選藥物的分子標的。在生物體中，NF-IL6和NFκB這兩個轉錄因子，在於免疫反應、發炎反應及細胞生長分化的調控上，皆扮演極為重要的角色，由於這些生理反應和人類的健康息息相關，所以我們分別將這兩個轉錄因子所能辨識的起動子區域與報導基因 ( reporter gene ) 做結合，而構築出一個可以方便應用於免疫、發炎及生長分化等相關藥物的篩選系統，我們所使用的報導基因分別為蟲螢光酵素 ( luciferase ) 及綠螢光蛋白 ( EGFP )。我們利用建構得的NF-IL6反應報導質體轉殖入HEK293細胞株並篩選得到了一株極為靈敏的細胞株，我們進一步利用staurosporine、前列腺素E1、PMA及forskolin來測試穩定保有報導基因的HEK293細胞株的蟲螢光素酵素的表現，發現20nM staurosporine在該細胞中可以對NF-IL6產生極大的刺激作用。作用8小時後酵素活性可達未刺激的20倍之多，此一結果與本實驗室近期的發現相吻合。PKC的活化劑PMA則在20小時後才見對NF-IL6有明顯的激發作用。前列腺素E1對NF-IL6的激發作用在4小時便可觀察到，並在8小時後達到最高峰；相類似的作用曲線亦見於adenylate cyclase 活化劑-forskolin的刺激上。此一結果說明前列腺素E1的刺激作用可能是經由活化adenylate cyclase 進而釋放cAMP的作用。我們目前正應用此一細胞株進行不同中草藥的粗萃液對前列腺素E1作用的觀察。未來我們將建立更多具有穩定表現報導基因的其他種類細胞株，相信會使系統更加的完備。

Transcription factor and the promoter ( or enhancer ) pair is one of generally used molecular markers for drug screening. Both NF-IL6 and NF-κB transcription factors, play an important role in regulation of many immune and inflammation responsive genes. These genes are normally coding for cytokines, cytokine receptors, cell adhesion molecules, and acute phase proteins. Hence, NF-IL6 and NF-κB are suitable markers for the screening of lead compounds for the regulation of immune and inflammation response. We frist constructed two reporter plasmids containing 3 sets NF-IL6 responding element ( p3X-NIL-LucNeo ) and 4 sets NF-κB responding elements ( p4X-NκB-LucNeo ) , respectively. The reporter plasmid p3X-NIL-LucNeo stablely transfected HEK293 cell clone was then generated and tested for its response to various agonists. We found that staurosporine could greatly induce luciferase activity of this cell clone in a time- and dose-dependent manner. The activity reached maximum at 50nM staurosporine of 14 hrs incubation. PMA, an activator of protein kinase C, on the other hand, resulted in a moderate stimulation on the NF-IL6-dependent luciferase expression after 20 hrs incubation. Interestingly, prostaglandinE1 induced a greater than 10-fold increase in luciferase activity compared with that of untreated cells. Forskolin, adenylate cyclase agonist, showed a similar stimulatory effect on the NF-IL6-dependent luciferase expression. The protein kinase A ( PKA ) –dependent phosphorylation and activation of NF-IL6 was postulated. In conclusion, we have develop a cell-based drug-screening system that may be useful in the searching of bio-active components in the traditional Chinses herbs. Here we show the generation and the characterization of HEK293 cell lines stably transfected with a reporter construct containing the firefly luciferase gene under the control of NF-IL6 or NFκB. And then we will make these lines potentially useful models for Chinese herb screening.