目的 利用正常人源肝細胞（HepaRG）和高內(nèi)涵技術(shù)檢測肝毒性標志物，并結(jié)合微核試驗和單細胞凝膠電泳試驗建立體外細胞毒性和遺傳毒性的快速篩選平臺。方法 選取適當?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HepaRG細胞活性氧簇（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和高通量彗星電泳試驗，綜合評價AQs致肝細胞毒性及染色體、DNA損傷情況。結(jié)果 與對照組比較，HepaRG細胞經(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)有文獻報道基本相符。本研究成功建立肝細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（2015ZX09501004-002）；中國食品藥品檢定研究院中青年發(fā)展研究基金（2014C1）