Luo S, Fang H Q, Yang H, Zhang L S, Jia X D
West China School of Public Health Sichuan University, Chengdu 610041, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2016 Jul 6;50(7):645-51. doi: 10.3760/cma.j.issn.0253-9624.2016.07.016.
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).
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, 250.0 μg/ml, and 500.0 μg/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, functions Ⅰ, Ⅱ and Ⅱ could be calculated by three linear discriminant functions in the EST model and the embryotoxicity of DEHP described by comparing the three functions.
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 246.8 μg/ml (ID50 D3). DEHP was evaluated to be a nonembryotoxic compound based on values of function Ⅰ (7.78), function Ⅱ (7.58) and function Ⅲ (-7.79). The three endpoints of DEHP in hEST were 195.4 μg/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.
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.
建立小鼠胚胎干细胞试验（mEST）模型和人胚胎干细胞试验（hEST）模型，以评估邻苯二甲酸二（2-乙基己基）酯（DEHP）的胚胎毒性。
我们根据欧洲替代方法验证中心（ECVAM）开发了mEST和hEST模型。分别使用青霉素G（PN-G）作为标准阴性对照物，5-氟尿嘧啶（5-FU）作为标准阳性对照物，以验证模型的有效性。基于模型有效性，对小鼠胚胎干细胞D3（mESC-D3）、小鼠Balb/c-3T3（3T3）和人胚胎干细胞H9（hESC-H9）给予不同浓度的DEHP（15.6、31.2、62.5、125.0、250.0、500.0和1000.0μg/ml），处理7天。使用细胞计数试剂盒-8检测DEHP对mESC-D3细胞、3T3细胞和hESC-H9细胞的50%抑制增殖浓度（IC50）。根据细胞毒性结果，用DEHP（15.6、31.2、62.5、125.0、250.0μg/ml和500.0μg/ml）处理mESC-D3和hESC-H9细胞10天。在第10天，通过实时PCR检测心肌细胞分化基因α-肌球蛋白重链（α-MHC）的表达，并确定50%心肌细胞分化抑制率（ID50）。根据IC50和ID50值，通过EST模型中的三个线性判别函数计算功能Ⅰ、Ⅱ和Ⅲ，并通过比较这三个函数来描述DEHP的胚胎毒性。
两种胚胎干细胞在最佳条件下培养，均高表达人胚胎干细胞标志物OCT4、SSEA4和TRA-1-60。形成的胚状体大小和形状均一，且这些结果具有高度可重复性。PN-G和5-FU的结果与ECVAM的预测一致。我们的EST模型验证结果令人满意。DEHP在mEST中的三个终点值分别为197.3μg/ml（3T3细胞的IC50）、210.0μg/ml（D3细胞的IC50）和246.8μg/ml（D3细胞的ID50）。根据功能Ⅰ（7.78）、功能Ⅱ（7.58）和功能Ⅲ（-7.79）的值，DEHP被评估为非胚胎毒性化合物。DEHP在hEST中的三个终点值分别为195.4μg/ml（3T3细胞的IC50）、184.8μg/ml（D3细胞的IC50）和84.3μg/ml（ID50）。通过比较功能Ⅰ（3.21）、功能Ⅱ（5.77）和功能Ⅲ（-6.46）的值，DEHP被评估为弱胚胎毒性。
mEST确定DEHP为非胚胎毒性化合物，hEST确定DEHP为弱胚胎毒性化合物。因此，hEST是评估DEHP胚胎毒性更敏感的模型。
