两种胚胎干细胞实验模型评价邻苯二甲酸（2-乙基己基）酯胚胎发育毒性的比较研究

Comparison of embryotoxicity of di（2-ethylhexyl） phthalate using mouse and human embryonic stem cell test models in vitro

目的：建立并应用小鼠胚胎干细胞实验（mEST）模型与人胚胎干细胞实验（hEST）模型评价邻苯二甲酸二（2-乙基己基）酯（DEHP）的胚胎发育毒性。方法参照欧洲替代方法研究中心（ECVAM）的标准操作方法，建立mEST、hEST模型，并选用ECVAM推荐的已知强胚胎发育毒性物质5-氟尿嘧啶（5-FU）和无胚胎毒性物质青霉素G（PN-G）进行模型验证，在模型验证有效的基础上，以15.6、31.2、62.5、125.0、250.0、500.0、1000.0μg/ml 浓度的DEHP分别染毒小鼠胚胎干细胞D3（mESC-D3）、Balb/c小鼠成纤维细胞3T3（3T3）及人胚胎干细胞H9（hESC-H9）7 d，采用CCK-8法检测不同浓度DEHP染毒后mESC-D3、3T3及hESC-H9细胞存活百分数和半数增殖抑制浓度（IC50）。根据细胞毒性实验结果，使用15.6、31.2、62.5、125.0、250.0、500.0μg/ml 浓度的DEHP分别处理分化的mESC-D3、hESC-H9细胞10 d，采用Real-time PCR检测不同浓度DEHP作用下，分化标志基因心肌细胞肌球蛋白重链（α-MHC）的表达情况，拟合效应曲线，得出半数分化抑制浓度（ID50）。并将ID50、IC50代入线性判别函数公式进行计算与比较。结果细胞生长状态良好，免疫荧光染色可见未分化hESC-H9细胞高表达、干性关键转录因子八聚体结合转录因子（OCT4）、阶段特异性胚胎抗原（SSEA-4）及肿瘤排斥抗原（Tra-1-60），细胞处于未分化状态。拟胚体大小均一、形态一致，可重复性高。采用ECVAM推荐已知发育毒性物质（5-FU和PN-G）对模型进行验证，所建模型有效，可用于受试物胚胎发育毒性的评价。DEHP对mESC-D3的IC50为210.0μg/ml、ID50为246.8μg/ml及对3T3的IC50为197.3μg/ml，代入线性判别函数公式得出函数Ⅰ、Ⅱ、Ⅲ值分别为7.78、7.58、-7.79，因Ⅰ〉Ⅱ〉Ⅲ，mEST模型评价DEHP为无胚胎发育毒性。DEHP对hESC-H9的IC50为184.8μg/ml，ID50为84.3μg/ml及对3T3的IC50为195.4μg/ml，代入线性判别函数公式得出函数Ⅰ、Ⅱ、Ⅲ值分别为3.21、5.77、-6.46，因Ⅱ〉Ⅰ〉Ⅲ， hEST模型评价DEHP为弱胚胎发育毒性。结论 hEST模型相对于mEST模型在评价受试物的弱胚胎发育毒性方面可能更为敏感。

Objective To establish a mouse embryonic stem cell test （mEST） model and human embryonic stem cell test （hEST） model, to evaluate the embryotoxicity of di（2-ethylhexyl） phthalate （DEHP）. Methods We developed mEST and hEST models according to the European Centre for the Validation of Alternative Methods （ECVAM）. We used penicillin G （PN-G） as the standard negative reference and 5-fluorouracil （5-FU） as the standard positive reference, respectively, to verify validity of the models. Based on model validity, mouse embryonic stem cells D3 （mESC-D3）, mouse Balb/c-3T3 （3T3）, and human embryonic stem cells H9 （hESC-H9） were administered different concentrations of DEHP （15.6, 31.2, 62.5, 125.0, 250.0, 500.0, and 1 000.0 μg/ml） for 7 days. A cell counting Kit-8 was used to detect the 50%inhibitory proliferation concentration （IC50） of mESC-D3 cells, 3T3 cells, and hESC-H9 with DEHP. mESC-D3 and hESC-H9 were treated with DEHP （15.6, 31.2, 62.5, 125.0, 250.0μg/ml, and 500.0μg/ml） for 10 days based on the cytotoxicity results. At day 10, the expression of cardiomyocyte differentiation gene alpha-myosin heavy chain （α-MHC） was detected by real-time PCR and the 50% inhibition of cardiomyocycte differentiation （ID50） determined. Based on the values of IC50 and ID50, functionsⅠ,ⅡandⅡcould be calculated by three linear discriminant functions in the EST model and the embryotoxicity of DEHP described by comparing the three functions. Results Nontrophoblast lineage both ES cells were cultured under optimal conditions and highly expressed hESC markers OCT4 , SSEA4, and TRA-1-60. The embryoid bodies formed were uniform in size and shape, and these results were highly repeatable. The PN-G and 5-FU results coincided with the prediction by ECVAM. Validation of our EST models was satisfactory. Results of the three endpoints of DEHP in mEST were 197.3 μg/ml （IC50 3T3）, 210.0 μg/ml （IC50 D3） and 246.8μg/ml （ID50 D3）. DEHP was evaluated to be a nonembryotoxic compound based on values of functionⅠ（7.78）, functionⅡ（7.58） and functionⅢ（-7.79）. The three endpoints of DEHP in hEST were 195.4μg/ml （IC50 3T3）, 184.8 μg/ml （IC50 D3）, and 84.3 μg/ml （ID50）. By comparing the values of function Ⅰ （3.21）, function Ⅱ （5.77）, and function Ⅲ （-6.46）, DEHP was evaluated to be weakly embryotoxic. Conclusion DEHP was determined to be a nonembryotoxic compound by mEST and weakly embryotoxic by hEST. Therefore, hEST is a more sensible model for the evaluation of DEHP embryotoxicity.