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, Nanning 530021, China.
Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
Zhongguo Zhong Yao Za Zhi. 2016 Apr;41(7):1313-1317. doi: 10.4268/cjcmm20160725.
3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line HepG2 and applied to evaluate the drug hepatotoxicity. After formation of stable 3D structure for HepG2 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 HepG2 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 HepG2 cells under 3D culture conditions were up-regulated 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 HepG2 cells showed higher sensitivity. This established 3D HepG2 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.
采用磁悬浮方法构建了人肝癌细胞系HepG2的三维体外毒性测试模型，并将其应用于评估药物肝毒性。在HepG2细胞形成稳定的三维结构后，采用免疫组织化学技术检测其在二维和三维培养条件下的糖原储存能力，并运用逆转录-聚合酶链反应（RT-PCR）方法比较二维和三维培养条件下Ⅰ相和Ⅱ相药物代谢酶、药物转运体、核受体以及肝脏特异性标志物白蛋白（ALB）的mRNA表达水平。免疫组织化学结果显示，HepG2细胞在三维培养条件下具有丰富的糖原储存能力，这与人肝脏组织相似。与二维培养条件相比，三维培养条件下HepG2细胞中主要药物代谢酶、药物转运体、核受体及ALB的mRNA表达水平上调。在药物肝毒性评估方面，选择典型的肝毒性药物对乙酰氨基酚（APAP）以及多数报道的药物何首乌和补骨脂进行单剂量和重复剂量（7天）暴露实验。在重复剂量暴露实验中，三维培养的HepG2细胞表现出更高的敏感性。这种采用磁悬浮三维培养技术建立的三维HepG2细胞模型在形态和功能上更接近人肝脏组织，因此是一种更好的三维肝毒性评估模型。
