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

Abstract

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

CLC Number:

X502

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.

References

[1] LUSTBERG M,SILBERGELD E. Blood lead levels and mortality[J]. Arch Intern Med,2002,162(21):2443-2449.
[2] AOKI Y,BRODY D J,FLEGAL K M,et al. Blood lead and other metal biomarkers as risk factors for cardiovascular disease mortality[J]. Medicine (Baltimore),2016,95(1):e2223.
[3] 陈侃,王长谦. 模式生物斑马鱼在心血管疾病研究中的应用[J]. 自然杂志,2013(1):64-72.
[4] DENIER C,LABAUGE P,BERGAMETTI F,et al. Genotype-phenotype correlations in cerebral cavernous malformations patients[J]. Ann Neurol,2006,60(5):550-556.
[5] HE Y,ZHANG H,YU L,et al. Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development[J]. Sci Signal,2010,3(116):a26.
[6] CHAN A C,DRAKOS S G,RUIZ O E,et al. Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice[J]. J Clin Invest,2011,121(5):1871-1881.
[7] FIDALGO M,GUERRERO A,FRAILE M,et al. Adaptor protein cerebral cavernous malformation 3(CCM3) mediates phosphorylation of the cytoskeletal proteins ezrin/radixin/moesin by mammalian Ste20-4 to protect cells from oxidative stress[J]. J Biol Chem,2012,287(14):11556-11565.
[8] 张晓. 中国水污染趋势与治理制度[J]. 中国软科学,2014(10):11-24.
[9] WESTERFIELD M. The Zebrafish Book[M]. Oregon:University of Oregon Press,1995.
[10] YORUK B,GILLERS B S,CHI N C,et al. CCM3 functions in a manner distinct from Ccm1 and Ccm2 in a zebrafish model of CCM vascular disease[J]. Dev Biol,2012,362(2):121-131.
[11] 曹梦思,陈锦瑶,张立实. 铅的心血管系统毒性研究进展[J]. 卫生研究,2014(6):1051-1056.
[12] JI A,WANG F,LUO W,et al. Lead poisoning in China:a nightmare from industrialisation[J]. Lancet,2011,377(9776):1474-1476.
[13] MEYER P A,BROWN M J,FALK H. Global approach to reducing lead exposure and poisoning[J]. Mutat Res,2008, 659(1/2):166-175.
[14] BARBOSA F J,TANUS-SANTOS J E,GERLACH R F,et al. A critical review of biomarkers used for monitoring human exposure to lead:advantages,limitations,and future needs[J]. Environ Health Perspect,2005,113(12):1669-1674.
[15] BOULDAY G,BLECON A,PETIT N,et al. Tissue-specific conditional CCM2 knockout mice establish the essential role of endothelial CCM2 in angiogenesis:implications for human cerebral cavernous malformations[J]. Dis Model Mech,2009, 2(3/4):168-177.
[16] MABLY J D,CHUANG L P,SERLUCA F C,et al. Santa and valentine pattern concentric growth of cardiac myocardium in the zebrafish[J]. Development,2006,133(16):3139-3146.
[17] STAHL S,GAETZNER S,VOSS K,et al. Novel CCM1, CCM2,and CCM3 mutations in patients with cerebral cavernous malformations:in-frame deletion in CCM2 prevents formation of a CCM1/CCM2/CCM3 protein complex[J]. Hum Mutat,2008,29(5):709-717.
[18] WHITEHEAD K J,PLUMMER N W,ADAMS J A,et al. CCM1 is required for arterial morphogenesis:implications for the etiology of human cavernous malformations[J]. Development, 2004, 131(6):1437-1448.
[19] ZAWISTOWSKI J S,STALHEIM L,UHLIK M T,et al. CCM1 and CCM2 protein interactions in cell signaling:implications for cerebral cavernous malformations pathogenesis[J]. Hum Mol Genet,2005,14(17):2521-2531.
[20] ZHANG J,RIGAMONTI D,DIETZ H C,et al. Interaction between krit1 and malcavernin:implications for the pathogenesis of cerebral cavernous malformations[J]. Neurosurgery, 2007,60(2):353-359.
[21] WIDZIOLEK M,PRAJSNAR T K,TAZZYMAN S,et al. Zebrafish as a new model to study effects of periodontal pathogens on cardiovascular diseases[J]. Sci Rep,2016,6:36023.
[22] PELSTER B,BURGGREN W W. Disruption of hemoglobin oxygen transport does not impact oxygen-dependent physiological processes in developing embryos of zebra fish (Danio rerio)[J]. Circ Res,1996,79(2):358-362.
[23] KALOGIROU S,MALISSOVAS N,MORO E,et al. Intracardiac flow dynamics regulate atrioventricular valve morphogenesis[J]. Cardiovasc Res,2014,104(1):49-60.
[24] MOULTON J D,YAN Y L. Using Morpholinos to control gene expression[J]. Curr Protoc Mol Biol,2008,26:26-28.
[25] 许嘉宁,陈燕. 我国水污染现状[J]. 广东化工,2014,41(3):143-144.
[26] 刘伟成,单乐州,谢起浪,等. 生物监测在水环境污染监测中的应用[J]. 环境与健康杂志,2008,25(5):456-459.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LIU Ruiguo, CHEN Xiaoli, YOU Yingbin, LI Shufan, ZHANG Qingying. Relationships between dyslipidemia and serum levels of lead and cadmium in low school-aged children in a district of Shenzhen [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(6): 461-465.
[2]	WANG Xiaohui, XIAO Chen, ZHAO Heping, WU Sai, XIA Dong, LIU Huaixiang, LIU Jieyi, ZHAO Zhiqiang, HE Yun. Exposure to low-level lead associated with glucose tolerance, retinal vascular permeability and diabetic retinopathy in mice [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2020, 32(2): 139-144.
[3]	LIU Mengya, SU Jiakun, WANG Ruoqi, XU Da, QIN Xiujun, LI Jianguo, AN Quan, CAI Jibao. Differential protein expressions in heart tissues from rats exposed to cigarette smoke [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(6): 444-448.
[4]	WU Desheng, WANG Honghui, YUAN Jianhui, WANG Hongju, ZHAO Qionghui, LIU Jianjun. Effect of fine particle exposure on development of zebrafish embryos [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(4): 307-309,314.
[5]	LU Yao, LIU Xinxia, XING Xiumei, ZHAO Zhiqiang, OU Xiaoyan, SU Xiaolin, SUN Yi, CHEN Jingli, SHEN Biling, JIANG Jun, HE Yun. DNA damage and telomere length changes in peripheral leukocytes from lead exposed workers [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(3): 179-184.
[6]	JIANG Jun, SUN Yi, LU Yao, ZHAO Zhiqiang, CHEN Jingli, ZHAO Heping, WU Sai, GAO Yanfang, XING Xiumei, HE Yun. Usefulness of primary mouse placental pericyte culture to screen for placental vascular toxicants [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(3): 209-213.
[7]	LI Xuling, WU Desheng, HONG Wenxu, LIU Jianjun, KE Yuebin, YUAN Jianhui, LI Shuangfei. Effects of nano-SiO₂ particles on zebrafish behavior response [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2015, 27(6): 467-471,476.
[8]	YIN Jian，WANG Ai-ping，LI Wan-fang，JIN Hong-tao，WEI Jin-feng. Establishment and application for an oxidative stress biomarker quantitative analysis system [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2014, 26(5): 321-329.
[9]	ZHU Min，ZHANG Qing-ying，LUO Jia-yi，ZHANG Dong-hong，ZHAO Zhi-guo，YI De-qing. The effect of lead and cadmium on mRNA of S100 calcium-binding protein family members in JAR cells [J]. , 2012, 24(3): 166-171.
[10]	CHEN Yan1; LIU Ji-wen1; ZHAO Jiang-xia1; CUI Jun2; LI Ling2; A Si-ya2; ZHU Yan3; TIAN Wei1. Polymorphisms of δ-Aminole- vulinic Acid Dehydratase; Vit D Receptor and Genetic Suscep- tibility of Lead Posioning in Han; Uygur and Kazak Children [J]. , 2009, 21(4): 280-285.
[11]	HAN Dai, XU Xi-jin, ZHANG Yu-qin, ZHENG Liang-kai, LI Yan, GU Cheng-wu, CHEN Gang-jian, CHEN Song-jian, LIU Jun-xiao,HUO Xi. Effect of Lead on Apoptosis and Expression of XIAP,Smac Genes in the Hippocampus of Developing Rats [J]. , 2008, 20(2): 119-122.
[12]	AN Lan-min1 ,NIU Yu-jie2 ,XU Bing2,LIU Na1. Influence of Lead on the Apoptosis and the Expression of fos，jun，p53 and Nitric Oxide Synthase in Rat Brain [J]. , 2006, 18(5): 359-362.
[13]	DONG Jie-ying1;JIN Long-jin1;LOU Zhe-feng1;ZHENG Zhong2;ZHAO Hui-ling3. Study of DNA Damage of Lead Acetate on Germ Cells of Mice in Vivo and in Vitro [J]. , 2006, 18(1): 42-045.
[14]	AN Lan - min1, YANG Jun1,XU Bing2. STUDIES ON THE APOPTOSIS AND THE EXPRESSION OF p53 BY LEAD IN RAT BRAIN [J]. , 2003, 15(4): 209-211.
[15]	Fu Shaolian , Li Hong. EFFECTS OF LEAD ON RAT EMBRYO MIDBRAIN CELL PROLIFERATION AND DIFFERENTIATION [J]. , 1999, 11(5): 249-251.