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中华介入放射学电子杂志 ›› 2022, Vol. 10 ›› Issue (04) : 429 -435. doi: 10.3877/cma.j.issn.2095-5782.2022.04.016

基础研究

血管内皮细胞Ddx24基因条件性敲除鼠构建以及对视网膜血管新生的影响
李冰1, 甘海润2, 蔡建勋2, 龙浩宇2, 李露婷2,()   
  1. 1. 519000 广东珠海,中山大学附属第五医院眼科;广东省生物医学影像重点实验室;广东省分子影像技术工程研究中心
    2. 中山大学附属第五医院介入医学中心;广东省生物医学影像重点实验室;广东省分子影像技术工程研究中心
  • 收稿日期:2022-05-10 出版日期:2022-11-25
  • 通信作者: 李露婷
  • 基金资助:
    国家自然科学基金(82072033); 广东省自然科学基金(2021A1515010380); 中山大学临床医学研究5010计划项目(2018011)

Establishment of a mouse model with Ddx24 conditional knock out in vascular endothelial cell and retina angiogenesis analysis

Bing Li1, Hairun Gan2, Jianxun Cai2, Haoyu Long2, Luting Li2,()   

  1. 1. Department of Ophthalmology,; Guangdong Provincial Key Laboratory of Biomedical Imaging,; Guangdong Provincial Engineering Research Center of Molecular Imaging, the Fifth Affiliated Hospital, Sun Yat-sen University, Guangdong Zhuhai 519000, China
    2. Center for Interventional Medicine,; Guangdong Provincial Key Laboratory of Biomedical Imaging,; Guangdong Provincial Engineering Research Center of Molecular Imaging, the Fifth Affiliated Hospital, Sun Yat-sen University, Guangdong Zhuhai 519000, China
  • Received:2022-05-10 Published:2022-11-25
  • Corresponding author: Luting Li
引用本文:

李冰, 甘海润, 蔡建勋, 龙浩宇, 李露婷. 血管内皮细胞Ddx24基因条件性敲除鼠构建以及对视网膜血管新生的影响[J]. 中华介入放射学电子杂志, 2022, 10(04): 429-435.

Bing Li, Hairun Gan, Jianxun Cai, Haoyu Long, Luting Li. Establishment of a mouse model with Ddx24 conditional knock out in vascular endothelial cell and retina angiogenesis analysis[J]. Chinese Journal of Interventional Radiology(Electronic Edition), 2022, 10(04): 429-435.

目的

构建血管内皮细胞Ddx24基因条件性敲除小鼠(Cdh5-Cre × Ddx24flox/flox),并利用该模型探索Ddx24基因对小鼠胚胎发育和视网膜血管新生的影响,为进一步探讨DDX24基因在内脏血管畸形中发挥的功能作用和具体分子机制提供动物模型。

方法

运用Cre-LoxP系统构建Ddx24基因的Flox修饰小鼠,并与血管内皮细胞特异性表达Cre酶的Cdh5-Cre小鼠进行繁育,以建立Cdh5-Cre ×Ddx24flox/flox小鼠模型。利用PCR和琼脂糖凝胶电泳进行基因鉴定,然后利用Western blot或qPCR实验验证Cdh5-Cre × Ddx24flox/flox小鼠血管组织中DDX24蛋白和mRNA的表达改变,最后通过H&E染色和免疫荧光实验观察Cdh5-Cre × Ddx24flox/flox小鼠胚胎和视网膜血管发育的变化。

结果

成功构建Cdh5-Cre × Ddx24flox/flox小鼠,并通过Western Blot和qPCR检测发现Cdh5-Cre × Ddx24flox/flox小鼠血管组织中DDX24表达显著下降;在血管内皮细胞中敲除Ddx24基因后无胚胎致死性,可导致幼鼠视网膜血管新生异常增多。

结论

利用Cre-LoxP系统获得Cdh5-Cre × Ddx24flox/flox小鼠,发现血管内皮细胞条件性敲除Ddx24基因可使幼鼠视网膜血管新生异常增多,为研究DDX24在内脏血管畸形中的作用提供了有效动物模型。

Objective

To establish vascular endothelial specific knockout of Ddx24 mice (Cdh5-Cre × Ddx24flox/flox) and explore the effect of Ddx24 gene on embryos development and retina neovascularization, so as to provide a model for investigating the functional role of DDX24 gene in visceral vascular malformation and specific molecular mechanism.

Methods

The flox-labeled mice with Ddx24 gene were established by using Cre-LoxP system and mated with Cdh5-Cre mice expressing Cre enzyme specifically in vascular endothelium for establishing Cdh5-Cre × Ddx24flox/flox mice. PCR and agarose gel electrophoresis were used for genotyping. Western blot or qPCR assay was used to observe the expression changes of DDX24 in vascular of Cdh5-Cre × Ddx24flox/flox. H&E-stained or immunofluorescence was used to observe the changes of embryos development and retina neovascularization in Cdh5-Cre × Ddx24flox/flox.

Results

Cdh5-Cre × Ddx24flox/flox were successfully established. Western blot and qPCR assay showed that the levels of DDX24 were decreased in the vascular tissue of Cdh5-Cre × Ddx24flox/flox. Vascular endothelial specific knockout of Ddx24 does not lead to embryonic death but result in abnormally increased retinal neovascularization.

Conclusions

The study successfully established Cdh5-Cre × Ddx24flox/flox mice using Cre-LoxP technique. Vascular endothelial specific knockout of Ddx24 could result in abnormally increased retinal neovascularization. Therefore, this animal model laid the foundation for exploring the functional role of DDX24 gene in visceral vascular malformation.

表1 基因鉴定引物信息
表2 PCR反应体系
图1 Ddx24flox/flox小鼠及血管内皮细胞Ddx24基因条件性敲除鼠构建示意图1A:Ddx24靶向载体和Ddx24flox等位基因构建示意图,2个LoxP序列位于3、5外显子两侧,与Cdh5-Cre小鼠繁育得到血管内皮细胞Ddx24基因条件性敲除鼠;1B:提取鼠尾DNA进行PCR和琼脂糖凝胶电泳,引物位置见1A。
图2 Cdh5-Cre × Ddx24flox/flox小鼠繁育策略及敲除效率验证2A:Cdh5-Cre × Ddx24flox/flox小鼠繁育策略;2B:小鼠基因型鉴定结果图,野生型为426 bp,Ddx24flox为518 bp,Cdh5-Cre为1 933 bp;2C:野生型和Cdh5-Cre × Ddx24flox/flox小鼠血管组织DDX24蛋白表达水平;2D:野生型和Cdh5-Cre × Ddx24flox/flox小鼠血管组织Ddx24 mRNA表达水平;****,P < 0.000 1。
图3 Cdh5-Cre × Ddx24flox/flox无胚胎致死性3A:E12 d野生型和Cdh5-Cre × Ddx24flox/flox小鼠胚胎图片(n = 3),比例尺为250 μm;3B:E12 d野生型和Cdh5-Cre × Ddx24flox/flox小鼠胚胎H&E染色图片(n = 3),比例尺为500 μm。
图4 Cdh5-Cre × Ddx24flox/flox小鼠视网膜血管异常增多4A:使用isolectin-IB4抗体染出生后第6天幼鼠视网膜组织并行共聚焦显微镜成像图片(n = 3),比例尺为100 μm;4B:视网膜血管密度统计图,*指P < 0.05。
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