基于胚胎干细胞模型的Cry1Ab蛋白发育毒性

doi:10.19723/j.issn.1671-167X.2024.02.003

摘要/Abstract

摘要：

目的: 通过胚胎干细胞发育毒性评价模型研究Cry1Ab蛋白对于细胞增殖和分化能力的影响，以评估其发育毒性。方法: 设置Cry1Ab蛋白7个剂量组(31.25、62.50、125.00、250.00、320.00、1 000.00、2 000.00 μg/L)，以5-氟尿嘧啶(5-fluorouracil，5-FU)为阳性对照，以磷酸缓冲盐溶液(phosphate buffer saline，PBS)为溶剂对照，分别处理小鼠胚胎干细胞D3(embryonic stem cell line D3，ES-D3)和小鼠成纤维细胞3T3。通过CCK-8法检测细胞活性，计算受试物对于不同细胞的增殖半抑制浓度(50% inhibition concentration of growth and viability，IC₅₀)。设置Cry1Ab蛋白5个剂量组(125.00、250.00、320.00、1 000.00、2 000.00 μg/L)，设置溶剂对照(PBS)，同时以5-FU为受试物进行模型验证，分别处理细胞后，通过拟胚胎体(embryonic bodies，EBs)培养法诱导ES-D3分化出心肌细胞；镜下观察EBs生长情况并测量其第3天和第5天的直径，观察并记录同批次EBs分化出搏动心肌细胞的比例，计算受试物的心肌分化半抑制浓度(50% inhibition concentration of differentiation，ID₅₀)，根据发育毒性判别函数对受试物的胚胎发育毒性进行分类；收集培养终点的EBs样本，进行实时定量聚合酶链式反应(real-time quantitative po-lymerase chain reaction，qPCR)，检测心肌分化相关标志物(Oct3/4、GATA-4、Nkx2.5和β-MHC)的mRNA表达情况。结果: 5-FU的IC_{50, 3T3}为46.37 μg/L，IC_{50, ES}为32.67 μg/L，ID_{50, ES}为21.28 μg/L，根据判别函数结果将5-FU分类为强胚胎毒性物质。不同浓度的Cry1Ab蛋白处理组的3T3细胞和ES-D3细胞活性与对照组相比差异均无统计学意义(P>0.05)。与对照组相比，Cry1Ab蛋白处理组分化第3天和第5天的EBs直径差异无统计学意义(P>0.05)，EBs形态也未见明显差异；不同浓度Cry1Ab蛋白处理组的心肌分化率与对照组相比差异无统计学意义(P>0.05)。5-FU使β-MHC、Nkx2.5和GATA-4的mRNA表达水平降低(P < 0.05)，且具有剂量依赖趋势(P < 0.05)，而与细胞多能性相关的标志物Oct3/4 mRNA表达水平则呈升高趋势(P < 0.05)；Cry1Ab蛋白处理组的成熟心肌标志物β-MHC、心肌早期分化标志物Nkx2.5和GATA-4、多能性相关标志物Oct3/4的mRNA表达水平与对照组相比差异均无统计学意义(P>0.05)。结论: 本实验模型中未观察到31.25~2 000.00 μg/L的Cry1Ab蛋白具有发育毒性。

关键词: Cry1Ab蛋白, 发育毒性, 心肌细胞, 细胞分化, 胚胎干细胞

Abstract:

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). Conclusion: No developmental toxicity of Cry1Ab protein at concentrations ranging from 31.25 to 2 000.00 μg/L was observed in this experimental model.

Key words: Cry1Ab protein, Developmental toxicity, Cardiac myocytes, Cell differentiation, Em-bryonic stem cells

中图分类号:

R155.5

简远志,王菲,尹宁,周若宇,王军波. 基于胚胎干细胞模型的Cry1Ab蛋白发育毒性[J]. 北京大学学报（医学版）, 2024, 56(2): 213-222.

Yuanzhi JIAN,Fei WANG,Ning YIN,Ruoyu ZHOU,Junbo WANG. Developmental toxicity of Cry1Ab protein in the embryonic stem-cell model[J]. Journal of Peking University (Health Sciences), 2024, 56(2): 213-222.

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Primer	Sequences (5′ to 3′)
β-MHC	Forward: CCTGCGGAAGTCTGAGAAGG
β-MHC	Reverse: CTCGGGACACGATCTTGGC
GATA-4	Forward: CACCCCAATCTCGATATGTTTGA
GATA-4	Reverse: GCACAGGTAGTGTCCCGTC
Nkx2.5	Forward: GACAAAGCCGAGACGGATGG
Nkx2.5	Reverse: CTGTCGCTTGCACTTGTAGC
Oct3/4	Forward: CGGAAGAGAAAGCGAACTAGC
Oct3/4	Reverse: ATTGGCGATGTGAGTGATCTG
GAPDH	Forward: CGTCCCGTAGACAAAATGGT
GAPDH	Reverse: TTGATGGCAACAATCTCCAC

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