探討干擾素及內毒素刺激誘導人類介白素12 p40 (IL-12 p40)基因轉錄作用分子機轉之研究

Abstract

介白素-12 ( Interleukin-12 )為抗原呈現細胞受到病原菌刺激後產生之細胞激素之一。主要之功能為活化自然殺手細胞及促使輔助型T細胞成熟。介白素12由p35及p40兩個次單元以雙硫鍵連接構成。多數細胞皆可表現p35次單元，而p40只在活化後的抗原呈現細胞中才大量表現。 我們選殖人類介白素12 p40啟動子區間-922至+35，此區間之序列經由軟體比對發現除了目前已知之轉錄因子辨識位置之外，仍有一些與免疫相關之轉錄因子辨識區域可能存在。我們將啟動子區域序列自5’端選擇性截斷消去，再將這些不同長度之啟動子區域序列置於蟲螢光酵素基因之前，來建構一系列的啟動區截斷型報導質體，以電穿孔方法將這些質體分別送入細胞中。在本研究中我們成功建立了穩定持有不同長度之人類介白素12 p40啟動子區域蟲螢光酵素報導基因的小鼠巨噬細胞株 (RAW264.7)，以此做為探討LPS/IFN-□以及中草藥初萃物分子機制之工具。我們發現LPS/IFN-□同時處理細胞可以協同性地激活啟動子區間-563至-398及Ets-2辨識區間(-222至-212)。後者為相關研究中常被提及之部分。經由軟體比對發現-433至-413□□□區間為c-Rel辨識區，以-433至-413之DNA序列為探針進行膠體位移實驗，結果顯示確實有蛋白質會結合至此區間。此外，我們也利用現有之報導質體來篩選具有活化介白素12 p40之中草藥初萃物並找出可能作用之啟動子區間。我們發現部分中草藥之萃取物確實能有效促進巨噬細胞株之介白素12 p40轉錄作用之進行。往後，我們將進一步分離出具有生物活性之前導化合物，並深入探討中草藥促進巨噬細胞介白素12 p40作用之分子機轉。

Interleukin-12 (IL-12) is a cytokine secreted by antigen-presenting cells (APCs) in response to pathogen infections. The major functions of IL-12 are to activate natural killer cells and promote naïve T helper cells to functional type 1 T helper cells. IL-12 is a heterodimeric cytokine composing of two distinct subunits, p35 and p40 linked by disulfide bonds. The p35 is constitutively expressing in most cells, whereas p40 is inducible and can only be detected in activated APCs. The promoter region -922 to +35 of human IL-12 p40 gene containing several putative transcription factor binding elements has been subcloned and their sequence alignment confirmed. A set of luciferase reporter plasmids containing various length of IL-12 p40 promoter were constructed and transfected into RAW264.7 cells to generate compounding stable cell clones. The stable clones of RAW264.7 containing human IL-12 p40 promoter□based reporter vectors were used to study the molecular mechanisms underlying LPS/IFN-□□ or Chinese herbs□ induced IL-12 p40 gene activation. We found that promoter regions -563 to -398 and -239 to -190 are important for the synergistic effect of LPS/IFN-□. The region -239 to -190 contains a Ets-2 (-222 to -212) site, which is consistent with previous finding. The result from gel shift assay, two bands were seen using region -433 to -413 as probe. Upon sequence alignment, a putative c-Rel binding element was postulated in region -563 to -398. Furthermore, the reporter plasmids pIL12p40/90 containing RAW cells were used to screen for the bioactive components of various Chinese Herbs. The result implicated that our system is useful for the future drugs screening and molecular mechanism studies of Chinese herbs.