丁香酚胚胎毒性评价:应用胚胎干细胞的实验模型

Embryotoxicity of eugenol based on a model of embryonic stem cell test

背景:丁香酚在医学及食品领域的应用越来越广泛,其药理学作用也不断被研究和发现。但是,对其毒性的研究尚未建立完整的数据库,特别是发育毒性及致畸性的安全性评价亟需完善。目的:建立胚胎干细胞实验模型的基础上,应用胚胎干细胞实验模型初步评价丁香酚的胚胎毒性。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和受试物丁香酚对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;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。利用胚胎毒性预测模型预测丁香酚的胚胎毒性。结果与结论:丁香酚对3T3细胞和E14TG2a细胞的增殖均有抑制作用,其半数生长抑制浓度值分别为(3.613±0.192)和(1.799±0.131)mg/L。丁香酚对E14TG2a细胞的心肌分化亦有抑制作用,其半数分化抑制浓度值为(3.501±0.158)mg/L。根据胚胎干细胞实验模型预测模型计算得出丁香酚为强胚胎毒性化合物。这为进一步评价丁香酚的安全性提供了研究数据。

BACKGROUND： As the pharmacological effect of eugenol constantly being discovered, its application in medical and food industry becomes wider. However, its toxicity studies have not established a complete database, especially in the improvement of safety assessment of developmental toxicity and teratogenicity. OBJECTIVE： To establish a model of embryonic stem cell test to evaluate the embryotoxicity of eugenol. METHODS： Mouse fibroblasts （3T3） and mouse embryonic stem cells （E14TG2a） were cultured in vitro, and MTT test was performed to detect the cytotoxicity of 3T3 cells and E14TG2a cells with positive control 5-fluorouracil, negative control penicillin G and tested compound eugenol. The concentration of the tested compounds that inhibiting 50% viability of embryonic stem cells （ICso 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 tested compounds that caused 50% inhibition of differentiation of E14TG2a cells into cardiomyocytes （ID50 E14TG2a） was calculated. The embryotoxic potential of eugenol was classified by prediction model of the embryonic stem cell test. RESULTS AND CONCLUSION： The proliferations of E14TG2a and 3T3 cells were inhibited by eugenol, of which the IC50 3T3 and IC50 E14TG2a values were （3.613±0.192） and （1.799±0.131） mg/L. The differentiationof E14TG2a was also inhibited by eugenol, of which the IDs0 E14TG2a was （3.501±0.158） mg/L. Eugenol was evaluated as a chemical compound with strong embryotoxicity by the model of embryonic stem cell test.