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目的应用胚胎干细胞(ESC)和胚胎干细胞试验(EST),初步评价多菌灵的胚胎毒性,并探讨可能的毒性机制。方法应用Alarma Blue法检测多菌灵对ESC和3T3成纤维细胞活性的影响;未分化的ESC体外经悬滴、悬浮培养后用10-4mol/L抗坏血酸诱导分化为具搏动能力的心肌细胞,暴露于不同浓度的多菌灵(0、1、2.5、5、10和20 nmol/L),在分化终点即第10天时获取分化细胞的RNA,并检测细胞多能性基因Oct4和心肌特异性基因α-MHC的相对表达量,计算多菌灵对ESC分化的半数抑制剂量。根据EST的评价标准,对多菌灵的胚胎毒性进行评价。结果多菌灵对3T3细胞和ESC细胞的存活率随着多菌灵浓度的增加而显著降低,ESC对多菌灵毒性更敏感;1 nmol/L多菌灵使Oct4的表达量显著升高,而2.5、5、10和20 nmol/L多菌灵使Oct4的表达量显著降低;随着多菌灵浓度的增加,α-MHC基因表达量逐渐降低,呈剂量-效应关系。多菌灵经EST评价为强胚胎毒性化合物。结论多菌灵具强胚胎毒性,其对ESC细胞活性及多能性基因影响显著可能是其作用的机制之一。
Objective To evaluate the embryotoxicity of carbendazim using embryonic stem cells (ESCs) and embryonic stem cell (EST) test and to explore the possible mechanism of toxicity. Methods Alarma Blue method was used to detect the effect of carbendazim on the activity of ESC and 3T3 fibroblasts. Undifferentiated ESCs were suspended in suspension in vitro. After suspension culture, cardiomyocytes were pulsed with 10-4mol / L ascorbic acid and exposed to At different concentrations of carbendazim (0, 1, 2.5, 5, 10, and 20 nmol / L), differentiated cells were harvested at the end of differentiation for 10 days and examined for cellular pluripotency genes Oct4 and cardiac muscle specific genes α-MHC relative expression levels, calculated carbendazim ESCs half-inhibitory dose. According to the evaluation criteria of EST, the carbendazim embryo toxicity was evaluated. Results Carbendazim survival rate of 3T3 cells and ESC cells decreased significantly with the increase of carbendazim concentration, and ESC was more sensitive to carbendazim toxicity. Oct4 expression was significantly increased by 1 nmol / L carbendazim, However, the expression of Oct4 was significantly decreased by 2.5, 5, 10, 10 and 20 nmol / L carbendazim. With the increase of carbendazim concentration, the expression of α-MHC gene gradually decreased, showing dose-effect relationship. Carbendazim was evaluated as a strong embryotoxic compound by EST. Conclusions Carbendazin has strong embryotoxicity and its effect on ESC cell activity and pluripotency genes may be one of the mechanisms.