中国中药杂志

2016, v.41(07) 1313-1317

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3D HepG2细胞药物肝毒性评价模型的建立及其在药物安全性评价中的应用
3D evaluation model for drug hepatotoxicity testing on HepG2 cells and its application in drug safety evaluation

李丹丹;汤响林;谭洪玲;梁乾德;王宇光;马增春;肖成荣;高月;
LI Dan-dan;TANG Xiang-lin;TAN Hong-ling;LIANG Qian-de;WANG Yu-guang;MA Zeng-chun;XIAO Cheng-rong;GAO Yue;Guangxi Medical University;Institute of Radiation Medicine,Academy of Military Medical Sciences;

摘要(Abstract):

该研究采用磁悬浮3维(3D)培养技术建立了3D Hep G2细胞评价模型,并应用此模型对典型的肝毒性药物进行了简单评价。Hep G2细胞形成稳定3D结构后,采用免疫组化技术检测了Hep G2细胞在2D,3D培养条件下的糖原储存能力,并应用RT-PCR技术对比研究了Hep G2细胞在2D,3D不同培养条件下Ⅰ相、Ⅱ相药物代谢酶、药物转运体、核受体及肝细胞特异标志性分子白蛋白(ALB)等相关基因的mRNA表达水平。免疫组化结果显示,Hep G2细胞在3D培养条件下具有丰富的糖原储存能力,此特性与人肝组织相类似。此外,在3D培养条件下,Hep G2细胞绝大部分药物代谢酶、转运体、核受体及ALB的mRNA表达水平与2D相比均有不同程度的上调。在药物肝毒性评价方面,该实验选用了典型的肝毒性药物对乙酰氨基酚(APAP)和近年来肝毒性报道较多的中药何首乌Polygonum multiflorum Thunb.及补骨脂Psoraleae corylifolia L.进行单剂量和重复剂量(7 d)给药实验;在重复剂量给药实验中,3D Hep G2细胞表现出更高的敏感性。应用磁悬浮3D培养技术建立的3D Hep G2细胞模型无论在形态上还是功能上都更接近于人的肝组织,是较好的3D肝毒性评价模型。
3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line Hep G2 and applied to evaluate the drug hepatotoxicity. After formation of stable 3D structure for Hep G2 cells,their glycogen storage capacity under 2D and 3D culture conditions were detected by immunohistochemistry technology,and the mRNA expression levels of phase Ⅰ and Ⅱ drug metabolism enzymes,drug transporters,nuclear receptors and liver-specific marker albumin( ALB) were compared between 2D and 3D culture conditions by using RT-PCR method. Immunohistochemistry results showed that Hep G2 cells had abundant glycogen storage capacity under 3D culture conditions,which was similar to human liver tissues. The mRNA expression levels of major drug metabolism enzymes,drug transporters,nuclear receptors and ALB in Hep G2 cells under 3D culture conditions were upregulated as compared with 2D culture conditions. For drug hepatotoxicity evaluation,the typical hepatotoxic drug acetaminophen( APAP),and most reported drugs Polygonum multiflorum Thunb.( Chinese name He-shou-wu) and Psoraleae corylifolia L.( Chinese name Bu-gu-zhi) were selected for single dose and repeated dose( 7 d) exposure. In the repeated dose exposure test,3D Hep G2 cells showed higher sensitivity. This established 3D Hep G2 cells model with magnetic levitation 3D culture techniques was more close to the human liver tissues both in morphology and functions,so it was a better 3D hepatotoxicity evaluation model.

关键词(KeyWords): 3D培养;肝毒性;评价模型
3D culture;hepatotoxicity;evaluation model

Abstract:

Keywords:

基金项目(Foundation): 北京市自然科学基金项目(7164291);; 国家“重大新药创制”科技重大专项(2014ZX09304307-001-003,2015ZX09501004-003-003);; 中医药行业科研专项(201507004)

作者(Authors): 李丹丹;汤响林;谭洪玲;梁乾德;王宇光;马增春;肖成荣;高月;
LI Dan-dan;TANG Xiang-lin;TAN Hong-ling;LIANG Qian-de;WANG Yu-guang;MA Zeng-chun;XIAO Cheng-rong;GAO Yue;Guangxi Medical University;Institute of Radiation Medicine,Academy of Military Medical Sciences;

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