论文部分内容阅读
目的探讨双酚A(BPA)对胚胎干细胞(ESC)分化潜能的影响,为合理评价BPA的安全性提供实验基础。方法制备及培养的小鼠胚胎成纤维细胞(MEF)和小鼠ESC,用BPA 0.1,1,10,100和1000μmol·L-1持续培养8 d,CCK-8法检测MEF和ESC细胞存活率并计算IC50;通过实时荧光定量PCR检测ESC中α肌球蛋白重链m RNA表达并计算其半数最大分化抑制浓度(ID50)。悬滴悬浮法培养的拟胚体,用BPA 0.001,0.01,0.1和1μmol·L-1持续培养10 d,实时荧光定量PCR检测拟胚体各胚层标志基因表达的变化。结果 BPA对小鼠ESC的IC50为5.22×10-4mol·L-1,对MEF的IC50为6.25×10-4mol·L-1,对小鼠ESC体外心肌细胞定向分化的ID50为7.0×10-7mol·L-1。BPA 0.001和0.01μmol·L-1可以上调拟胚体的中胚层标志基因胎肝激酶1和球蛋白转录因子1的表达。结论 BPA属于强胚胎毒性化合物。低浓度BPA对小鼠ESC分化为中胚层细胞有促进分化的作用。
Objective To investigate the effect of BPA on the differentiation potential of embryonic stem cells (ESC) and provide experimental basis for evaluating the safety of BPA. Methods Mouse embryonic fibroblasts (MEFs) and mouse ESCs were prepared and cultured. The cells were cultured continuously with BPA 0.1, 1, 10, 100 and 1000μmol·L-1 for 8 days. The survival rates of MEF and ESC cells were determined by CCK-8 assay IC50. The expression of α-myosin heavy chain m RNA in ESCs was detected by real-time fluorescence quantitative PCR and the half maximum inhibitory concentration (ID50) was calculated. Embryoid bodies cultured in suspension suspension method were cultured with BPA at the concentrations of 0.001, 0.01, 0.1 and 1 μmol·L-1 for 10 days. Real-time quantitative PCR was used to detect the expression of the marker gene in embryonic germ layers. Results The IC50 of BPA for mouse ESCs was 5.22 × 10-4 mol·L-1, the IC50 of MEF was 6.25 × 10-4 mol·L-1, and the ID50 of mouse ESCs differentiated into cardiomyocytes was 7.0 × 10- 7mol · L-1. BPA 0.001 and 0.01μmol·L-1 could up-regulate the expression of mesodermal marker hepatic kinase 1 and globulin 1 in embryoid-derived embryos. Conclusion BPA is a strong embryo toxic compound. Low concentration of BPA can promote the differentiation of mouse ESCs into mesoderm cells.