基于拟胚体面积的体外胚胎毒性模型的建立和验证

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/3T3 5 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_{50 3T3}、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（ID₅₀）。根据得到的 IC_{50 3T3}、IC_{50 D3}、ID₅₀，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_{50 3T3}、IC_{50 D3}、ID₅₀，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＝21.550 926 58×lg IC_{50 3T3}－7.600 241 83×lg IC_{50 D3}－14.007 571 16×lg ID₅₀＋6.675 517 2，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_{50 3T3}、IC_{50 D3}、ID₅₀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。

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

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_{50 3T3}, 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 (ID₅₀). Then a linear discriminant analysis (LDA) was used to obtain the estimated linear discriminant function by Python based on the obtained IC_{50 3T3}, IC_{50 D3}, ID₅₀. Model verification: The IC_{50 3T3}, IC_{50 D3}, and ID₅₀ of ascorbic acid C, ibuprofen and cisplatin were obtained by the same method as above. The function score were calculated by the prediction model.Result The estimated linear discriminant function is: discriminant value = 21.550 926 58×lg IC_{50 3T3}-7.600 241 83×lg IC_{50 D3}-14.007 571 16×lg ID₅₀+6.675 517 2, 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.