用于筛选脑出血相关基因的斑马鱼模型的构建  

Construction of zebrafish models for screening intracranial hemorrhage associated genes

作　　者：闫思宇 张秀茹 张仁杰 马潞[1] 李浩[1] 贺民[1] 伍聪[1] 肖安琪[1] 游潮[1] 刘翼[1] 王业启 田蕊 Yan Siyu;Zhang Xiuru;Zhang Renjie;Ma Lu;Li Hao;He Min;Wu Cong;Xiao Anqi;You Chao;Liu Yi;Wang Yeqi;Tian Rui(Department of Neurosurgery,West China Hospital,Sichuan University,Chengdu 610041,China;Bioengineering College,Chongqing University,Chongqing 400030,China)

机构地区：[1]四川大学华西医院神经外科,成都610041 [2]重庆大学生物工程学院,重庆400030

出　　处：《中华医学杂志》2022年第33期2619-2623,共5页National Medical Journal of China

基　　金：国家重点研发计划(2018YFA0108603,2018YFA0108604);四川大学华西医院学科卓越发展1·3·5工程临床研究孵化项目(2018HXFH007,2021HXFH014);华西医院院-企合作临床研究创新项目(2019HXCX07)。

摘　　要：目的构建斑马鱼脑出血模型,并以此筛选诱导斑马鱼脑出血的相关基因。方法通过吗啉代寡核苷酸(MO)技术和单细胞显微注射技术构建斑马鱼脑出血模型,并从宏观、微观角度多重验证。首先,造模实验采用注射Control MO的正常野生型AB品系斑马鱼作为对照组,实验组在AB斑马鱼胚胎单细胞时期分别注射神经嵴源性细胞(NCDCs)发育相关基因的MO,包括col8a1 MO、tfap2αMO、msx1a MO、msx2 MO、dkk1a MO等,于显微镜白光视野下初步验证模型。其次,采用Tg(flk1:GFP;gata1:dsRed)双转基因斑马鱼进行模型验证,该转基因鱼用绿色荧光蛋白(GFP)标记血管内皮细胞,用红色荧光蛋白(dsRed)标记血红细胞,能够更清晰地观察到斑马鱼脑出血的部位。在斑马鱼胚胎单细胞时期注射Control MO作为对照组,注射col8a1 MO作为实验组,正常培养至受精后48 h通过激光共聚焦观察红细胞渗漏情况,构建荧光视野可观察的脑出血模型。最后,采用Tg(flk1:GFP)转基因斑马鱼进行基于血脑屏障的模型验证,通过Dextran-Rhodamin和DAPI染料的渗漏现象进一步明确斑马鱼血脑屏障的破坏与脑出血的发生,并进行定量统计,从而验证NCDCs发育相关基因与脑出血表型的关系,证明造模有效。结果与注射Control MO的正常野生型AB品系斑马鱼对照组相比,col8a1、tfap2α和msx1基因突变的实验组斑马鱼有明显的脑出血现象,出血比例分别为18.18%(52/286)、23.04%(62/251)、35.94%(23/64),而msx2和dkk1a基因突变的斑马鱼极少观察到脑出血,出血比例仅分别为1.03%(1/97)和1.15%(1/87)。注射Control MO与col8a1 MO的Tg(flk1:GFP;gata1:dsRed)双转基因斑马鱼红细胞渗漏发生率分别为0.37%(1/273)与18.18%(52/286)(P<0.001)。注射Control MO与col8a1 MO组Tg(flk1:GFP)转基因斑马鱼DAPI阳性的细胞核数分别为(10.05±5.27)个与(60.35±3.96)个(P<0.001);Dextran-Rhodamin染料渗漏引起的中脑实质荧光示踪强度值分别为2.54±4.7Objective To construct zebrafish models for the screening of intracranial hemorrhage(ICH)associated genes.Methods ICH zebrafish models were constructed through morpholino oligonucleotides(MOs)technique and microinjection technique,and multiple verification was performed from macro and micro perspectives.First,the normal wild-type AB strain zebrafish injected with control MO was used as the control group,and AB zebrafish embryos microinjected with MOs of genes related to development of neural crest-derived cells(NCDCs)were used as the study group,such as col8a1 MO,tfap2αMO,msx1a MO,msx2 MO,and dkk1a MO.Preliminary verification of the model was conducted under a white-light optical microscope.Then,the model was verified by Tg(flk1:gfp;gata1:dsRed)double transgenic zebrafish,with vascular endothelial cells labeled by green fluorescent protein(GFP)and red blood cell labeled by fluorescent protein(dsRed),and thus the location of cerebral hemorrhage can be observed more clearly.Specifically,zebrafish embryos were microinjected with Control MO as the control group and those microinjected with col8a1 MO as the study group.Then the embryos were cultured until 48 hours post-fertilization to observe the leakage of red blood cells under the confocal laser scanning microscope.Finally,Tg(flk1:gfp)transgenic zebrafish was used to verify the model based on the blood-brain barrier(BBB).Through the leakage of dextran-rhodamine and DAPI dyes,the destruction of BBB and the occurrence of cerebral hemorrhage in zebrafish were further clarified,and quantitative statistics were carried out to verify the relationship between NCDCs development related genes and cerebral hemorrhage phenotype,which proved that the modeling was effective.Results The zebrafish with col8a1,tfap2α,and msx1 mutations in the study group had apparent ICH compared with wildtype zebrafish,and the prevalence of ICH was 18.18%(52/286),23.04%(62/251),and 35.94%(23/64),respectively.While,the zebrafish with msx2 and dkk1a mutations rarely had ICH,with the ICH prevalenc

关 键 词：脑出血 斑马鱼 血脑屏障 渗漏 基因 

分 类 号：R743.34[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]