药学学报, 2021, 56(2): 503-510
引用本文:
毋姗姗, 范文娟, 李瑞玲, 王艳丽, 李培全, 李超杰, 邓锦波. 利用诱导多能干细胞构建流产毒性药物的体外筛查模型[J]. 药学学报, 2021, 56(2): 503-510.
WU Shan-shan, FAN Wen-juan, LI Rui-ling, WANG Yan-li, LI Pei-quan, LI Chao-jie, DENG Jin-bo. Construction of a drug screening model for miscarriage from induced pluripotent stem cells in vitro [J]. Acta Pharmaceutica Sinica, 2021, 56(2): 503-510.

利用诱导多能干细胞构建流产毒性药物的体外筛查模型
毋姗姗1,2, 范文娟3, 李瑞玲2, 王艳丽1, 李培全1, 李超杰1, 邓锦波1
1. 河南省生殖健康科学技术研究院, 国家卫健委出生缺陷预防重点实验室, 河南省人口缺陷干预技术研究重点实验室, 河南 郑州 450002;
2. 河南大学护理与健康学院, 河南 开封 475000;
3. 漯河医学高等专科学校, 河南 漯河 462002
摘要:
妊娠期用药不可避免,如何帮助孕妇合理用药以减少自发流产、早产和低体重儿的发生,对于医务工作者至关重要。本研究建立了药物流产毒性的检测模型,可筛除疑似流产的药物,避免用药不当导致的孕妇流产。通过三维悬浮培养诱导多能干细胞(induced pluripotent stem cells,iPSCs)的方式,诱导其形成拟胚体(embryoid bodies,EBs)和类皮质器官,在体外模拟早期胚胎从内细胞团向三胚层体系的分化过程。根据流产药物米非司酮(RU486)暴露浓度的不同,随机将EBs分为3组:空白对照组(control)、低浓度组(L-RU486,10 μg·mL-1)和高浓度组(H-RU486,20 μg·mL-1)。借助末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法[terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling,TUNEL]和免疫荧光染色等技术观察第5、8和11天的不同组别EBs的大小、细胞凋亡和胚层分化的情况。研究结果表明:iPSCs体外可聚集成三维结构的EBs,在特异性神经诱导因子作用下,EBs外周分化出神经玫瑰花状结构,形成大脑皮质类器官;米非司酮暴露导致EBs发育迟缓,EBs直径减少(P<0.01),促进EBs内部细胞凋亡(P<0.01),并影响EBs胚层发育,抑制胚胎干细胞增殖和外胚层细胞分化(P<0.01),促进中胚层细胞发育(P<0.05)。本研究提示,iPSCs可作为药物流产毒性的筛选模型,而EBs的大小、细胞凋亡率和胚层细胞分化率可作为药物筛选的评价指标。
关键词:    诱导多能干细胞      生殖发育毒性      模型建立      药物筛选     
Construction of a drug screening model for miscarriage from induced pluripotent stem cells in vitro
WU Shan-shan1,2, FAN Wen-juan3, LI Rui-ling2, WANG Yan-li1, LI Pei-quan1, LI Chao-jie1, DENG Jin-bo1
1. National Health Commission Key Laboratory of Birth Defects Prevention, Henan Key Laboratory of Population Defects Prevention, Henan Institute of Reproduction Health Science and Technology, Zhengzhou 450002, China;
2. School of Nursing and Health, Henan University, Kaifeng 475000, China;
3. Luohe Medical College, Luohe 462002, China
Abstract:
Drug use during pregnancy is unavoidable. Therefore, it is vitally important for medical workers to help pregnant women take drugs correctly to reduce the incidence of spontaneous abortion, premature birth, and low birth weight. In our study, drug screening model with induced pluripotent stem cells (iPSCs) was used to find some improper drugs which will result in woman's abortion. With 3D culture in vitro, iPSCs can form embryoid bodies (EBs) and cerebral organoids, which simulated in vitro development of early embryos, from inner cell mass to germ-layer differentiation. In the experiment, EBs were exposed to mifepristone (RU486), and three experimental groups were divided randomly. They were control group (without RU486), low-dose group (L-RU486, 10 μg·mL-1), and high-dose group (H-RU486, 20 μg·mL-1). After mifepristone exposure, EBs were observed at days 5, 8, and 11, including size of EB, cell apoptosis, and differentiation of germ layers, by using inverted optical microscope, TUNEL assay, and immunofluorescent staining. The results showed that through 3D culture, iPSCs could develop into embryoid bodies, neural rosettes, and finally cerebral organoids. After mifepristone exposure, EBs' sizes were decreased (P<0.01); the levels of cell apoptosis in EBs were increased after mifepristone exposure (P<0.01); the development of EBs' germ layer was affected. Mifepristone exposure could inhibit the proliferation of embryonic stem cells, reduce the differentiation of ectoderm (P<0.01) and promote the development of mesoderm (P<0.05). In conclusion, iPSCs can be used as a screening model for abortion drug, and EBs' diameter, cell apoptosis, and differentiation changes of the germ layers can serve as criteria of abortion drug screening.
Key words:    induced pluripotent stem cell    reproductive and developmental toxicity    model establishment    drug screening   
收稿日期: 2020-09-21
DOI: 10.16438/j.0513-4870.2020-1508
基金项目: 国家卫健委出生缺陷预防重点实验室开放课题(ZD201903);河南省科技厅科技攻关项目(192102310134);河南省医学科技攻关计划(联合共建)项目(LHGJ20190827,LHGJ20190828).
通讯作者: 邓锦波,Tel:13613788951,Fax:86-371-65961650,E-mail:jinbo_deng2017@126.com
Email: jinbo_deng2017@126.com
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