利用体外替代模型评价栀子黄色素的发育毒性

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摘要目的利用胚胎干细胞试验、大鼠植入后全胚胎培养试验和微团培养试验评价栀子黄色素的胚胎发育毒性。方法分别检测栀子黄色素对小鼠胚胎干细胞ES-D3及胚胎成纤维细胞BALB/3T3增殖的影响,并检测栀子黄色素对ES-D3细胞向心肌细胞分化能力的影响;分离孕9.5 d大鼠胚胎进行全胚胎培养,测量胚胎生长指标,并根据Brown′s评分法对胚胎进行总形态学评分;分离孕13 d大鼠胚胎肢芽细胞进行微团培养,利用中性红活细胞摄取染色法测定细胞增殖情况,利用阿利新蓝染色法观察细胞分化情况。根据欧洲替代方法验证中心(European Centre for the Validation of Alternative Methods, ECVAM)的胚胎干细胞试验、全胚胎培养及微团培养预测模型对栀子黄色素的发育毒性进行综合评价。结果栀子黄色素对ES-D3细胞和BALB/3T3细胞增殖活性的最大无作用水平(no observed adverse effect level, NOAEL)分别为31.25 μg/mL和125 μg/mL,IC₅₀-_D3和IC₅₀-_3T3分别为231.87 μg/mL和236.57 μg/mL;其对ES-D3向心肌细胞分化能力影响的NOAEL为31.3 μg/mL,ID₅₀为94.84 μg/mL;1 000 μg/mL栀子黄色素对胚胎生长及形态发育无明显影响;栀子黄色素对肢芽细胞增殖活性及向软骨细胞分化能力的NOAEL均为50 μg/mL,其IC₅₀和ID₅₀分别为175 μg/mL和152 μg/mL。结论经胚胎干细胞试验模型和微团培养模型评价栀子黄色素为弱胚胎发育毒性化学物;经全胚胎培养预测模型评价栀子黄色素为无胚胎早期发育毒性化学物。

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	康陈萍
	肖倩倩
	刘青云
	郝卫东

关键词 ：栀子黄, 胚胎干细胞, 全胚胎培养, 微团培养, 发育毒性

Abstract：Objective To assess the potential developmental embryonic toxicity of gardenia yellow with embryonic stem cell test, post-implantation whole embryo culture test and micromass test.Methods In embryonic stem cell test, the effect of gardenia yellow on proliferation of mouse embryonic stem cell ES-D3 and embryonic fibroblast BALB/3T3, as well as differentiation of ES-D3 into cardiomyocytes, were tested. In post-implantation whole embryo culture test, the rat embryos at day 9.5 of gestation were derived and cultured. The growth index of embryos were measured and the total morphological score were calculated according to Brown′s Method. In micromass test, the limb bud cells of embryos were isolated from mature rats at day 13 of gestation and cultured. The proliferation of limb bud cells was determined by neutral red staining and the differentiation was measured by alcian blue staining. The developmental toxicity of gardenia yellow was evaluated with the prediction models of European Centre for the Validation of Alternative Methods (ECVAM).Results The no observed adverse effect level (NOAEL) of gardenia yellow on proliferation of ES-D3 and BALB/3T3 was 125 μg/mL and 31.25 μg/mL, the IC_50-D3 and IC_50-3T3was 236.57 μg/mL and 231.87 μg/mL, respectively. The NOAEL of gardenia yellow on differentiation ability of ES-D3 into cardiomyocytes was 31.3 μg/mL,and the ID₅₀ was 94.84 μg/mL。Gardenia yellow had no significant effect on embryo growth and morphology in 1 000 μg/mL. The NOAEL of gardenia yellow on both proliferation and differentiation of limb bud cell was 50 μg/ml, the IC₅₀ and ID₅₀were 175 μg/mL and 152 μg/mL, respectively.Conclusion Gardenia yellow was evaluated as weak embryonic developmental toxic substance by embryonic stem cell test and micromass test prediction models, and as non-embryogenic toxic substance by post-implantation whole embryo culture prediction model.

Key words： Gardenia yellow Embryonic stem cell test Whole embryo culture Micromass Test Developmental toxicity.

收稿日期: 2022-01-18

ZTFLH:	R11
	R17

基金资助:国家重点研发计划(2017YFC1600203)

通讯作者: 肖倩倩(xqq1017@foxmail.com)

引用本文:

康陈萍, 肖倩倩, 刘青云, 郝卫东. 利用体外替代模型评价栀子黄色素的发育毒性[J]. 中国生育健康杂志, 2022, 33(3): 247-253. KANG Chenping, XIAO Qianqian, LIU Qingyun, HAO Weidong. Assessment of developmental toxicity of gardenia yellow pigment by in vitro alternative models. Chinese Journal of Reproductive Health, 2022, 33(3): 247-253.

链接本文:

http://cjrh.bjmu.edu.cn/CN/ 或 http://cjrh.bjmu.edu.cn/CN/Y2022/V33/I3/247

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