藉由轉錄調控因子與三維支架誘導人類皮膚纖維母細胞表現 cytochrome P450 以建立藥物代謝之預測平台

Abstract

準確有效且方便的藥物代謝與毒性預測之篩選平台對於新藥開發是非常重要，然而目前卻仍然缺乏。本研究首先選用24個對於肝臟再生與發育相當重要的轉錄因子 (transcription factors) 與核受體 (nuclear receptors)，並最後篩選出10個轉錄調控因子，能誘導人類皮膚纖維母細胞 (human dermal fibroblasts, HDFs) 表現 phase I 藥物代謝的重要酵素：cytochrome P450 (CYP) 3A4、1B1 與 2C9。誘導後的人類皮膚纖維母細胞之CYPs的基因表現量與活性均高於目前普遍使用的肝癌細胞株 (hepatoma cell lines)。藉由將誘導後的細胞培養成球體或將細胞種入不同類型的三維細胞支架材料，尤其是由明膠 (gelatin)、軟骨素 (chondroitin) 和透明質酸 (hyaluronan) 所組成的三共聚物材料可以更進一步的增加CYPs的基因表現量與活性。值得注意的是，誘導後的細胞培養成球體並種入三共聚物材料對於增加CYPs的基因表現量與活性具有顯著的加乘效應。掃描式電子顯微鏡和共軛焦顯微鏡顯示，這些細胞球體能穩定的存在三維細胞支架材料內部並且展現高度存活率。此外，球體之細胞培養於三維細胞支架材料中，具有能將高血壓藥物-硝苯地平 (nifedipine) 代謝成氧化硝苯地平 (oxidized nifedipine) 之能力，顯示此平台可適用於藥物代謝之研究。本研究提出了一項新策略，能誘導人類皮膚纖維母細胞表現phase I 藥物代謝的重要酵素之基因且具有活性，未來將有助於建立體外藥物代謝的研究平台可用於預測藥物對人體的毒性。

A reliable, reproducible, and convenient in vitro platform for drug metabolism determination and toxicity prediction is of tremendous value but still lacking. In the present study, a collection of 24 hepatic transcription factors and nuclear receptors in different combinations were surveyed, and 10 among them were finally selected to induce the expression and enzyme activities of cytochrome P450 (CYP) 3A4, 1B1, and 2C9 in human dermal fibroblasts (HDFs). The expression and activities of these CYPs in the induced HDFs were higher than those in commonly used hepatoma cell lines. Even higher CYP expression and activities could be further enhanced by culturing the induced HDFs either as spheroids or into several kinds of scaffolds, particularly the tri-copolymer scaffold composed of gelatin, chondroitin, and hyaluronan. More strikingly, there showed a synergistic effect of seeding and culturing the spheroids into the tri-copolymer scaffold. Scanning electron microscopy and confocal microscopy disclosed well accommodation of these spheroids inside the scaffolds and displayed a high survival rate. Moreover, the spheroid/scaffold constructs could metabolize an anti-hypertension drug nifedipine into oxidized nifedipine, showing their applicability in studying drug metabolism. This study presents a novel strategy to induce the expression and enzyme activities of critical CYPs in HDFs, and may have potential to establish an in vitro platform to study drug metabolism and to predict the possible human risk of drug toxicity.