CCM3基因敲降斑马鱼模型的建立及其在毒理学中的初步应用

doi:10.3969/j.issn.1004-616x.2017.03.007

癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (3): 194-198,204.doi: 10.3969/j.issn.1004-616x.2017.03.007

CCM3基因敲降斑马鱼模型的建立及其在毒理学中的初步应用

申碧羚¹, 郭小玲², 何云¹

1. 广州市环境污染与健康评价重点实验室, 中山大学公共卫生学院预防医学系, 广东广州 510080;
2. 中山大学附属第一医院, 广东广州 510080

收稿日期:2017-02-23 修回日期:2017-04-21 出版日期:2017-05-31 发布日期:2017-05-31
通讯作者: 何云,E-mail:heyun7@mail.sysu.edu.cn E-mail:heyun7@mail.sysu.edu.cn
作者简介:申碧羚,E-mail:565575720@qq.com。
基金资助:
国家自然科学基金（81273097，81472998）；中山大学“百人计划”引进人才科研启动基金

Establishment of a CCM3 gene knockdown zebrafish model and its preliminary application in toxicology

SHEN Biling¹, GUO Xiaoling², HE Yun¹

1. Guangzhou Key Laboratory of Environmental Pollution and Risk Assessment, School of Public Health, Sun Yat-sen University, Guangzhou 510080;
2. The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong, China

Received:2017-02-23 Revised:2017-04-21 Online:2017-05-31 Published:2017-05-31

摘要/Abstract

摘要： 目的：建立CCM3基因敲降的斑马鱼模型，探讨其作为水体中低浓度心血管毒性污染物的生物监测模型的可能性。方法：选取绿色荧光标记的血管内皮细胞生长因子受体2（VEGFR2）转基因斑马鱼作为研究对象，采用吗啡啉修饰的反义寡聚核苷酸（MO）对斑马鱼单细胞期受精卵进行显微注射，采用激光共聚焦显微镜观察受精后72 h的幼鱼脑部血管发育形态，建立CCM3基因敲降的斑马鱼模型。通过改进寇氏法测定铅对斑马鱼的半数致死剂量（LD₅₀）。在低浓度10 mg/L铅暴露下分别对正常基因型和CCM3基因敲降型的斑马鱼卵进行染毒，同时设立对照组（无铅暴露），观察各组斑马鱼发育情况。结果：野生型斑马鱼铅染毒的LD₅₀为31.24 mg/L。在相同低浓度10 mg/L铅暴露的情况下，受精后72 h时正常基因型的斑马鱼幼鱼和对照组的斑马鱼幼鱼各项发育指标无明显异常，而注射亚表型剂量的CCM3 MO 0.25 ng组斑马幼鱼出现形态发育异常表型，如发育迟缓、脊柱弯曲、心包水肿、卵黄囊水肿等。结论：CCM3基因敲降的斑马鱼模型可作为水体中具有心血管毒性污染物的敏感生物监测模型。

关键词: CCM3基因, 铅, 斑马鱼, 心血管系统, 生物监测

Abstract: OBJECTIVE:To establishment a zebrafish model with CCM3 gene knockdown and to investigate its usefulness as a biological monitoring model for low concentrations of cardiovascular toxicants in water. METHODS:The transgenic zebrafish line (VEGFR2:GFP) which expresses green flurescence proteins in blood vessels were used. The one-cell stage zebrafish eggs were injected with morpholino oligos (MO) at indicated doses. Laser scanning confocal microscopy was used to observe the intracranial vessels of zebrafish at 72 h. The zebrafish model with CCM3 gene knockdown was established. Karber's method was used to calculate the median lethal dose (LD₅₀) of lead for zebrafish. The wild-type and CCM3 gene knockdown zebrafish were exposed to low concentrations of lead (10 mg/L). Development of zebrafish of each group was documented. RESULTS:The LD₅₀ value for wild-type zebrafish was 31.24 mg/L. Zebrafish with CCM3 gene knockdownt were more susceptible to low concentrations of lead-exposure (10 mg/L) than the wild-type by displaying abnormal phenotype,such as decreased body length,pericardial edema,hemorrhage,egg yolk edema, spine distortions. Up to 72 h,after treatment with the low dose,the wild-type zebrafish did not show any abnormal phenotype. CONCLUSION:The CCM3 gene knockdown zebrafish can possibly be used as a sensitive biological monitoring model for cardiovascular toxicants in water.

Key words: CCM3gene, lead, zebrafish, cardiovascular system, biological monitoring

中图分类号:

X502

申碧羚, 郭小玲, 何云. CCM3基因敲降斑马鱼模型的建立及其在毒理学中的初步应用[J]. 癌变·畸变·突变, 2017, 29(3): 194-198,204.

SHEN Biling, GUO Xiaoling, HE Yun. Establishment of a CCM3 gene knockdown zebrafish model and its preliminary application in toxicology[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(3): 194-198,204.

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CCM3基因敲降斑马鱼模型的建立及其在毒理学中的初步应用

Establishment of a CCM3 gene knockdown zebrafish model and its preliminary application in toxicology

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