目的 利用小鼠胚胎干細(xì)胞，以擬胚體面積為分化抑制終點(diǎn)，構(gòu)建體外胚胎毒性模型。方法 模型建立：分別用訓(xùn)練集10個(gè)化合物青霉素G、異煙肼、5-氟尿嘧啶、羥基脲、阿司匹林、氟康唑、地塞米松、丙戊酸鈉、洛伐他汀、苯海拉明處理小鼠胚胎干細(xì)胞ES-D3和小鼠胚胎成纖維細(xì)胞NIH/3T3 5 d，用細(xì)胞活力檢測(cè)試劑盒檢測(cè)細(xì)胞活性，得到相應(yīng)的半數(shù)抑制濃度（IC_{50 3T3}、IC_{50 D3}）；同時(shí)用10種化合物處理ES-D3細(xì)胞，用懸浮-懸滴培養(yǎng)的途徑制備擬胚體，于第5天對(duì)擬胚體進(jìn)行拍照，用 Image J 軟件測(cè)量擬胚體的橫截面積，并計(jì)算半數(shù)抑制細(xì)胞分化的濃度（ID₅₀）。根據(jù)得到的 IC_{50 3T3}、IC_{50 D3}、ID₅₀，用Python軟件采用線性判別分析（LDA）擬合得到線性二分類判別式。模型驗(yàn)證：以上述同樣的方法測(cè)定測(cè)試集3個(gè)化合物維生素C、布洛芬、順鉑的IC_{50 3T3}、IC_{50 D3}、ID₅₀，將其代入得到的判別式，并預(yù)測(cè)化合物的胚胎毒性。結(jié)果 通過訓(xùn)練集 10個(gè)化合物擬合出線性預(yù)測(cè)判別式為：判別值＝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，判別值＜0，歸為無胚胎毒性；判別值≥0，歸為胚胎毒性。將測(cè)試集化合物的 IC_{50 3T3}、IC_{50 D3}、ID₅₀代入判別式，得到維生素C判別值＜0，為無胚胎毒性藥物；順鉑和布洛芬判別值＞0，為胚胎毒性藥物，均與文獻(xiàn)中體內(nèi)試驗(yàn)結(jié)果一致，預(yù)測(cè)正確。結(jié)論 基于擬胚體面積成功建立體外胚胎毒性模型。

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_{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.

R965.3

中國食品藥品檢定研究院中青年發(fā)展研究基金（2020C1）