® 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."/>

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首頁 > 過刊瀏覽>2017年第40卷第11期 >2017,40(11):1550-1558. DOI:10.7501/j.issn.1674-6376.2017.11.006
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人源HepaRG肝細(xì)胞毒性與遺傳毒性高通量篩選方法的初步建立

Preliminary validation of high throughput screening methods for hepatotocity and genotoxicity in humanized HepaRG cells

發(fā)布日期:2017-11-15
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