目的 利用正常人源肝細(xì)胞（HepaRG）和高內(nèi)涵技術(shù)檢測肝毒性標(biāo)志物，并結(jié)合微核試驗(yàn)和單細(xì)胞凝膠電泳試驗(yàn)建立體外細(xì)胞毒性和遺傳毒性的快速篩選平臺(tái)。方法 選取適當(dāng)?shù)臒晒馓结楬oechst33342、DCFH-DA、Fluo4-AM、MitoTracker^® Red CMX Ros聯(lián)合高內(nèi)涵技術(shù)研究不同大黃蒽醌類單體（AQs）對(duì)HepaRG細(xì)胞活性氧簇（ROS）、胞內(nèi)Ca²⁺含量及線粒體膜完整性等肝毒性標(biāo)志物的影響，并開展高內(nèi)涵法胞質(zhì)分裂阻斷法微核試驗(yàn)和高通量彗星電泳試驗(yàn)，綜合評(píng)價(jià)AQs致肝細(xì)胞毒性及染色體、DNA損傷情況。結(jié)果 與對(duì)照組比較，HepaRG細(xì)胞經(jīng)25.0 μg/mL大黃素、12.5和25.0 μg/mL蘆薈大黃素、50和25.0 μg/mL大黃酚處理24 h后，胞內(nèi)ROS含量顯著增多；12.5和25.0 μg/mL蘆薈大黃素和50.0 μg/mL大黃酸可引起胞內(nèi)Ca²⁺含量顯著增多；大黃素25.0 μg/mL、蘆薈大黃素25.0 μg/mL、大黃酚50.0和25.0 μg/mL、大黃酸50.0和25.0 μg/mL組導(dǎo)致線粒體明顯損傷（P<0.05、0.01）。與對(duì)照組比較，25.0 μg/mL大黃素誘導(dǎo)微核率、尾DNA含量和彗星尾距（OTM）數(shù)值均顯著升高（P<0.05、0.01）；50.0 μg/mL大黃酚給藥72 h后微核率顯著升高（P<0.01）。結(jié)論 AQs的研究結(jié)果與現(xiàn)有文獻(xiàn)報(bào)道基本相符。本研究成功建立肝細(xì)胞毒性和遺傳毒性的聯(lián)合快速篩選模型，有助于藥物研發(fā)早期的毒性篩選。

Objective To detect the hepatotoxicity biomarkers using normal human hepatocyte (HepaRG) and high-content screening, and to combine the micronucleus test and single cell gel electrophoresis to estalish a rapid screening platform for in vitro cytotoxitity and genotoxicity. Methods The effects of rhubarb anthraquinones (AQs) on the reactive oxygen species (ROS), intracellular Ca²⁺ concentration and mitochondrial membrane potential (MMP) in HepaRG cells were studied using appropriate fluorescent probes Hoechst33342、DCFH-DA、Fluo4-AM、Mito Tracker Red CMX Ros and high-content screening methods, and the potential genotoxiciy triggered by AQs were analyzed using the high-content based cytokinesis block micronucleus test and high throughput comet assay. Results The intracellular ROS level of HepaRG cells was significantly elevated by a 24 h treatment with Emodin (25.0 μg/mL), aloe-emodin (25.0 μg/mL) or chrysophanol (50.0 μg/mL), which are dose-concentration dependent (P < 0.05 and 0.01); the intracellular Ca²⁺ increased and mitochondrial damage were observed with the treatment of aloe-emodin (25.0 μg/mL) and rhein (50.0 μg/mL, P < 0.05 and 0.01). Comparing to control group, Emodin (25.0 μg/mL) induced an increased micronucleus rate (1.59% ±0.68%,P < 0.01) and significantly higher percentage tail DNA and Olive tail moment (respectively 10.155% ±2.17% and 0.510 ±0.06, P < 0.05 and 0.01) after 24 h; while the chrysophanol increased the micronucleus rate to 1.29% ±0.54% (P < 0.01) after 72 h. Conclusion The results on the cytotoxicities and genotoxicities of AQs are consistent with the literatures. In this study, a rapid screening model for both hepatotoxicity and genotoxicity was successfully established, which will help with the early screening during the drug development stage.

國家"重大新藥創(chuàng)制"科技重大專項(xiàng)（2015ZX09501004-002）；中國食品藥品檢定研究院中青年發(fā)展研究基金（2014C1）