目的:采用基于胚胎干细胞的发育毒性评价模型,研究Cry1Ab蛋白对细胞增殖和分化能力的影响,评价其发育毒性。
方法:在小鼠胚胎干细胞D3(ES-D3)和3T3小鼠成纤维细胞上测试了七个剂量组(31.25、62.50、125.00、250.00、320.00、1000.00和2000.00μg/L)的Cry1Ab蛋白,用5-氟尿嘧啶(5-FU)作为阳性对照,用磷酸盐缓冲盐水(PBS)作为溶剂对照。通过CCK-8测定检测细胞活力以计算测试物质对不同细胞的50%抑制浓度(IC50)。此外,在ES-D3细胞上测试了五个剂量组(125.00、250.00、320.00、1000.00和2000.00μg/L)的Cry1Ab蛋白,PBS作为溶剂对照,5-FU用于模型验证。细胞处理后,使用胚胎体(EBs)培养方法诱导心脏分化。在显微镜下观察EB的生长,并在第三天和第五天测量它们的直径。记录分化为搏动心肌细胞的EB比例,并计算分化的50%抑制浓度(ID50)。基于发育毒性判别函数,对试验物质的发育毒性进行了分类。此外,在文化时期结束时,使用实时定量聚合酶链反应(qPCR)在收集的EB样品中定量检测心脏分化相关标志物(Oct3/4,GATA-4,Nkx2.5和β-MHC)的mRNA表达水平。
结果:在3T3细胞中测定5-FU的IC50为46.37μg/L,在ES-D3细胞中测定为32.67μg/L,ES-D3细胞的ID50为21.28μg/L。根据判别函数结果,5-FU被分类为强胚胎毒性物质。在3T3细胞和ES-D3细胞中,不同浓度的Cry1Ab蛋白处理组与对照组的细胞活力差异均无统计学意义(P>0.05)。此外,第3天和第5天的EBs直径无统计学差异,以及它们的形态,Cry1Ab蛋白处理组与对照组比较(P>0.05)。不同浓度Cry1Ab蛋白治疗组与对照组的分化率比较差异无统计学意义(P>0.05)。5-FU显著降低β-MHC的mRNA表达水平,Nkx2.5和GATA-4(P<0.05),呈剂量依赖性趋势(P<0.05),而多能性相关标志物Oct3/4的mRNA表达水平呈现增加趋势(P<0.05)。然而,成熟心脏标志物β-MHC的mRNA表达水平差异无统计学意义,早期心脏分化标志物Nkx2.5和GATA-4以及多能性相关标志物Oct3/4在Cry1Ab蛋白治疗组与对照组之间(P>0.05)。
结论:在该实验模型中,未观察到浓度范围为31.25至2000.00μg/L的Cry1Ab蛋白的发育毒性。
OBJECTIVE: To evaluate the developmental toxicity of Cry1Ab protein by studying its effects on cell proliferation and differentiation ability using a developmental toxicity assessment model based on embryonic stem-cell.
METHODS: Cry1Ab protein was tested in seven dose groups (31.25, 62.50, 125.00, 250.00, 320.00, 1 000.00, and 2 000.00 μg/L) on mouse embryonic stem cells D3 (ES-D3) and 3T3 mouse fibroblast cells, with 5-fluorouracil (5-FU) used as the positive control and phosphate buffer saline (PBS) as the solvent control. Cell viability was detected by CCK-8 assay to calculate the 50% inhibitory concentration (IC50) of the test substance for different cells. Additionally, Cry1Ab protein was tested in five dose groups (125.00, 250.00, 320.00, 1 000.00, and 2 000.00 μg/L) on ES-D3 cells, with PBS as the solvent control and 5-FU used for model validation. After cell treatment, cardiac differentiation was induced using the embryonic bodies (EBs) culture method. The growth of EBs was observed under a microscope, and their diameters on the third and fifth days were measured. The proportion of EBs differentiating into beating cardiomyocytes was recorded, and the 50% inhibition concentration of differentiation (ID50) was calculated. Based on a developmental toxicity discrimination function, the developmental toxicity of the test substances was classified. Furthermore, at the end of the culture period, mRNA expression levels of cardiac differentiation-related markers (Oct3/4, GATA-4, Nkx2.5, and β-MHC) were quantitatively detected using real-time quantitative polymerase chain reaction (qPCR) in the collected EBs samples.
RESULTS: The IC50 of 5-FU was determined as 46.37 μg/L in 3T3 cells and 32.67 μg/L in ES-D3 cells, while the ID50 in ES-D3 cells was 21.28 μg/L. According to the discrimination function results, 5-FU was classified as a strong embryotoxic substance. There were no statistically significant differences in cell viability between different concentrations of Cry1Ab protein treatment groups and the control group in both 3T3 cells and ES-D3 cells (P>0.05). Moreover, there were no statistically significant differences in the diameter of EBs on the third and fifth days, as well as their morphology, between the Cry1Ab protein treatment groups and the control group (P>0.05). The cardiac differentiation rate showed no statistically significant differences between different concentrations of Cry1Ab protein treatment groups and the control group (P>0.05). 5-FU significantly reduced the mRNA expression levels of β-MHC, Nkx2.5, and GATA-4 (P < 0.05), showing a dose-dependent trend (P < 0.05), while the mRNA expression levels of the pluripotency-associated marker Oct3/4 exhibited an increasing trend (P < 0.05). However, there were no statistically significant differences in the mRNA expression levels of mature cardiac marker β-MHC, early cardiac differentiation marker Nkx2.5 and GATA-4, and pluripotency-associated marker Oct3/4 between the Cry1Ab protein treatment groups and the control group (P>0.05).
CONCLUSIONS: No developmental toxicity of Cry1Ab protein at concentrations ranging from 31.25 to 2 000.00 μg/L was observed in this experimental model.