以NF-κB活性為基準之指標性基因測試法偵測中藥之內毒素污染   全文下載

Detection of Endotoxin Contamination in Chinese Herbs by NF-κB Activity-Based Reporter Assays

食品及藥物中嚴重的內毒素污染不僅對健康有威脅，有時也造成相關科學研究的錯誤結論。為了確保傳統中藥材的品質，其內毒素的污染問題應被審慎評估。本文介紹一種以NF-κB活性為基準之指標性基因測試法來偵測中藥之內毒素污染。我們首先建立了一個帶有指標性基因的特殊巨噬細胞株。此指標性基因含有NF-κB-responding sites及luciferase基因。當LPS存在時會活化NF-κB，進而引發下游luciferase基因的表現。經由加入LPS抑制劑polymyxin B可確認這些由LPS誘發的luciferase活性具有專一性。在我們的系統中，LPS含量與其所誘發的luciferase活性有極高的關連性，而LPS所誘發的luciferase活性可被polymyxin B完全抑制，偵測LPS的靈敏度為1 ng／mL。經由RT-PCR實驗我們也驗證了這個濃度下（1 ng／mL）已足以引起發炎相關細胞素TNF-α及IL-6的表現。我們進而利用此系統測試三十五種中藥材中內毒素的污染情形，我們所測試的中藥材約五分之一呈現不同程度的、可偵測的內毒素污染（高於1 ng／mL） 。這個結果顯示中藥材的內毒素污染是值得重視的問題。此外，此偵測法操作方便、低背景值等特性提供了其應用於系統化偵測中藥材內毒素污染的可能性。

Severe LPS contamination in drugs and food can cause health problems and occasionally mislead research conclusions. To ensure the quality of traditional Chinese medicinal herbs (TCMH), LPS contamination problem should be evaluated. We here described a NF-κB activity-based reporter assay to detect LPS contamination. We first created a macrophage cell line with integrated reporter gene consisting of NF-κB-responding sites upstream of the luciferase gene. The presence of LPS leads to NF-κB activation, and thus triggers downstream luciferase expression. The specificity of LPS-derived luciferase activity was confirmed by adding the LPS inhibitor, polymyxin B. In our system, the level of LPS correlates well with luciferase activity. The LPS activity is completely inhibited by polymyxin B, and the limit of LPS detection is 1ng/mL. We also utilized RT-PCR to demonstrate that LPS contamination at the concentration of 1ng/mL was enough to induce expression of downstream inflammatory cytokines TNF-a and IL-6. We further applied the method to examine LPS contamination in TCMH. Among the 35 herbal extracts we examined, about 20% of them exhibited variable but detectable LPS contamination (higher than 1ng/mL). The data indicate that LPS contamination in Chinese herbs should be considered. In addition, the ease and low-background feature of this assay suggest its potential application for systematic detection of LPS contamination in Chinese herbs. 食品及藥物中嚴重的內毒素污染不僅對健康有威脅，有時也造成相關科學研究的錯誤結論。為了確保傳統中藥材的品質，其內毒素的污染問題應被審慎評估。本文介紹一種以NF-κB活性為基準之指標性基因測試法來偵測中藥之內毒素污染。我們首先建立了一個帶有指標性基因的特殊巨噬細胞株。此指標性基因含有NF-κB-responding sites及luciferase基因。當LPS存在時會活化NF-κB，進而引發下游luciferase基因的表現。經由加入LPS抑制劑polymyxin B可確認這些由LPS誘發的luciferase活性具有專一性。在我們的系統中，LPS含量與其所誘發的luciferase活性有極高的關連性，而LPS所誘發的luciferase活性可被polymyxin B完全抑制，偵測LPS的靈敏度為1 ng／mL。經由RT-PCR實驗我們也驗證了這個濃度下（1 ng／mL）已足以引起發炎相關細胞素TNF-α及IL-6的表現。我們進而利用此系統測試三十五種中藥材中內毒素的污染情形，我們所測試的中藥材約五分之一呈現不同程度的、可偵測的內毒素污染（高於1 ng／mL） 。這個結果顯示中藥材的內毒素污染是值得重視的問題。此外，此偵測法操作方便、低背景值等特性提供了其應用於系統化偵測中藥材內毒素污染的可能性。