基于拟胚体面积的体外胚胎毒性模型的建立和验证  

Establishment and validation of an in vitro embryotoxicity model based on embryoid body's area

作　　者：赵曼曼 郑锦芬 黄芝瑛[2] 耿兴超[1] 张曦[1] 刘晓萌[1] 王三龙[1] 周晓冰[1] ZHAO Manman;ZHENG Jinfen;HUANG Zhiying;GENG Xingchao;ZHANG Xi;LIU Xiaomeng;WANG Sanlong;ZHOU Xiaobing(Beijing Key Laboratory,National Center for Safety Evaluation of Drugs,National Institutes for Food and Drug Control,Beijing 100176,China;School of Pharmaceutical Sciences,Sun Yat-Sen University,Guangzhou 510006,China)

机构地区：[1]中国食品药品检定研究院国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京100176 [2]中山大学药学院,广东广州510006

出　　处：《药物评价研究》2023年第5期925-933,共9页Drug Evaluation Research

基　　金：中国食品药品检定研究院中青年发展研究基金(2020C1)。

摘　　要：目的利用小鼠胚胎干细胞,以拟胚体面积为分化抑制终点,构建体外胚胎毒性模型。方法模型建立:分别用训练集10个化合物青霉素G、异烟肼、5-氟尿嘧啶、羟基脲、阿司匹林、氟康唑、地塞米松、丙戊酸钠、洛伐他汀、苯海拉明处理小鼠胚胎干细胞ES-D3和小鼠胚胎成纤维细胞NIH/3T35 d,用细胞活力检测试剂盒检测细胞活性,得到相应的半数抑制浓度(IC_(503T3)、IC_(50 D3));同时用10种化合物处理ES-D3细胞,用悬浮-悬滴培养的途径制备拟胚体,于第5天对拟胚体进行拍照,用Image J软件测量拟胚体的横截面积,并计算半数抑制细胞分化的浓度(ID_(50))。根据得到的IC_(503T3)、IC_(50 D3)、ID_(50),用Python软件采用线性判别分析(LDA)拟合得到线性二分类判别式。模型验证:以上述同样的方法测定测试集3个化合物维生素C、布洛芬、顺铂的IC_(503T3)、IC_(50 D3)、ID_(50),将其代入得到的判别式,并预测化合物的胚胎毒性。结果通过训练集10个化合物拟合出线性预测判别式为:判别值=21.55092658×lg IC_(503T3)-7.60024183×lg IC_(50 D3)-14.00757116×lg ID_(50)+6.6755172,判别值<0,归为无胚胎毒性;判别值≥0,归为胚胎毒性。将测试集化合物的IC_(503T3)、IC_(50 D3)、ID_(50)代入判别式,得到维生素C判别值<0,为无胚胎毒性药物;顺铂和布洛芬判别值>0,为胚胎毒性药物,均与文献中体内试验结果一致,预测正确。结论基于拟胚体面积成功建立体外胚胎毒性模型。Objective To establish an in vitro embryotoxicity model based on mouse embryonic stem cell-derived embryoid bodies.Method Model establishment:Mouse embryonic stem cells and NIH/3T3 cells were treated with penicillin G,isoniazid,5-fluorouracil,hydroxyurea,aspirin,fluconazole,dexamethasone,sodium valproate,lovastain,and diphenhydramine for 5 d,cell activity was detected with cell viability detection kit to obtain half inhibitory concentration(IC_(503T3),IC_(50 D3)).Embryoid bodies made by suspension-hanging method were photographed on day 5.The areas of embryoid bodies were measured by Image J and calculate the concentration of half inhibitory cell differentiation(ID_(50)).Then a linear discriminant analysis(LDA)was used to obtain the estimated linear discriminant function by Python based on the obtained IC_(503T3),IC_(50 D3),ID_(50).Model verification:The IC_(503T3),IC_(50 D3),and ID_(50) of ascorbic acid C,ibuprofen and cisplatin were obtained by the same method as above.The function score were calculated by the prediction model.ResultThe estimated linear discriminant function is:discriminant value=21.55092658×lg IC_(503T3)-7.60024183×lg IC_(50 D3)-14.00757116×lg ID_(50)+6.6755172,if the discriminant value<0,it is classified as nontoxic drug,otherwise,it is classified as toxic drug.The discriminant value of ascorbic acid C<0,the discriminant value of ibuprofen and cisplatin>0.Ascorbic acid C was classified as nontoxic drug,ibuprofen and cisplatin were classified as toxic drug,which were consistent with the classification by in vivo research.Conclusion An in vitro embryotoxicity model was established based on embryoid body area.

关 键 词：小鼠胚胎干细胞 拟胚体面积 胚胎毒性 线性判别分析 

分 类 号：R965.3[医药卫生—药理学]