应用胚胎干细胞实验模型评价三聚氰胺的胚胎毒性

Embryonic stem cell test for the study of the embryotoxicity of Melamine

目的建立胚胎干细胞实验模型,应用胚胎干细胞实验模型初步评价三聚氰胺的胚胎毒性。方法体外培养小鼠成纤维细胞3T3和小鼠胚胎干细胞E14TG2a,MTT法检测阳性对照5-氟尿嘧啶、阴性对照青霉素G和受试物三聚氰胺对3T3细胞和E14TG2a细胞的细胞毒性,计算3种化合物对E14TG2a细胞和3T3细胞的半数生长抑制浓度值IC50 3T3、IC50 E14TG2a;采用悬滴-悬浮-贴壁法体外诱导胚胎干细胞向心肌细胞分化,根据浓度-反应曲线计算3种化合物对E14TG2a细胞的半数分化抑制浓度值ID50 E14TG2a。利用胚胎毒性预测模型预测三聚氰胺的胚胎毒性。结果三聚氰胺对3T3细胞和E14TG2a细胞的增殖均有抑制作用,其IC50 3T3和IC50 E14TG2a分别为485.6±25.05和304.1±20.50μg/ml。三聚氰胺对E14TG2a细胞的分化亦有抑制作用,其ID50 E14TG2a为152.6±12.62μg/ml。根据EST预测模型计算得出三聚氰胺为弱胚胎毒性。结论三聚氰胺为弱胚胎毒性。

Objective： To establish a model of embryonic stem cell test （EST） to evaluate the embryotoxicity of Melamine. Methods： Mouse fibroblasts （3T3） and mouse ES cells （E14TG2a） were cultured in vitro, and MTT test was performed to detect the cytotoxicity of 3T3 cells and E14TG2a ceils with positive control 5-fluorouracih negative control penicillin G and tested compound Melamine. The concentration of the test compounds that inhibiting 50% viability of ES cells （IC5 E14TG2a） and 3T3 fibroblasts （IC50 3T3） was calculated. The hanging-suspension-adherent culture systems were used to induce embryonic stem cells into cardiomyocytes, and the concentration of test compounds that caused 50% inhibition of differentiation of E14TG2a ceils into cardiomyocytes （ID5014TG2a） was calculated. The embryotoxic potential of Melamine was classified by prediction model （PM） of the EST. Results： The proliferation of E14TG2a and 3T3 cells were inhibited by Melamine, of which the IC50 3T3 and IC50 E14TG2a values were 485.6±25.05 and 304.1±20.50μg/ml. The differentiation of E14TG2a was also inhibited by Melamine, of which the ID50E14TG2a was 152.6±12.62 μg/ml. Melamine was evaluated as a weak embryotoxicity chemical by EST.Conclusion： Melamine exhibited a weak embryotoxicity.