中国口腔颌面外科杂志 ›› 2018, Vol. 16 ›› Issue (1): 6-11.doi: 10.19438/j.cjoms.2018.01.002

• 论著 • 上一篇    下一篇

TIE2-R849W突变斑马鱼模型的建立及其在静脉畸形发病中的作用探讨

杜仲, 马海龙, 张凌, 郑家伟, 王延安   

  1. 上海交通大学医学院附属第九人民医院·口腔医学院 口腔颌面-头颈肿瘤科, 上海市口腔医学重点实验室,上海 200011
  • 收稿日期:2017-09-30 修回日期:2017-11-18 出版日期:2018-01-20 发布日期:2018-02-11
  • 通讯作者: 王延安,E-mail:wangyan_an@163.com
  • 作者简介:杜仲(1992-),男,硕士,E-mail:duzhongd987@163.com
  • 基金资助:
    国家自然科学基金(81371164)

Establishment of zebrafish model via TIE2-R849W and exploration of its role in hereditary venous malformations

DU Zhong, MA Hai-long, ZHANG Ling, ZHENG Jia-wei, WANG Yan-an   

  1. Department of Oromaxillofacial Head and Neck Oncology, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Stomatology. Shanghai 200011, China
  • Received:2017-09-30 Revised:2017-11-18 Online:2018-01-20 Published:2018-02-11

摘要: 目的: 建立TIE2-R849W突变斑马鱼模型并初步探讨其在静脉畸形中可能的致病机制。方法: 通过合成TIE2-R849W突变基因表达体系,利用成熟的mRNA显微注射技术,基于fli1a: EGFP转基因斑马鱼构建TIE2-R849W过表达斑马鱼模型,荧光显微镜下记录斑马鱼尾静脉丛(caudal vein plexus,CVP)的早期发育改变并定量比较。提取斑马鱼RNA,针对目标基因行实时定量PCR(qRT-PCR),利用已有基因芯片进一步验证结论。采用GraphPad Prism 5.0进行统计学分析。结果: 基于对TIE2-R849W过表达斑马鱼模型的观察,TIE2-R849W显著影响斑马鱼早期尾静脉丛的发育,表现为尾静脉丛窦腔数量和面积显著减少。qRT-PCR显示,TIE2-R894W可引发pik3r2foxo1b的表达上调,而与尾静脉丛发育相关的基因,除显著高表达的egfl7外,nr2f2nr2f1as1pr1均无显著异常表达。比较体外芯片结果,部分突变组内的tie2pik3r2foxo1begfl7同样存在一致的高表达。结论: TIE2-R849W引发斑马鱼尾静脉丛发育异常与egfl7的高表达关系密切。

关键词: 静脉畸形, 尾静脉丛, 斑马鱼, Egfl7

Abstract: PURPOSE: To explore the role of TIE2-R849W, a mutation related to hereditary venous malformations, via establishing a novel zebrafish model. METHODS: Through the designment and synthesis of TIE2-R849W expression plasmid, translated mRNA was microinjected into fli1a: EGFP fertilized eggs, which were used to establish a TIE2-R849W over-expression model. Under fluorescence microscope, the developmental change of caudal vein plexus (CVP) was recorded and quantitatively compared. Using RNA extracted from zebrafish, target genes were tested by real-time quantitative PCR (qRT-PCR). The existing gene chip was analyzed to further verify the results. Statistical analysis was performed with GraphPad Prism 5. RESULTS: Based on observed phenotypes of TIE2-R849W over-expression zebrafish, TIE2-R849W significantly affected the development of the caudal venous plexus, which displayed a significant decrease in the number and area of sinus. According to the results of qRT-PCR, upregulation of pik3r2 and foxo1b could be induced by TIE2-R894W over-expression. At the same time, except for significantly up-regulated egfl7, expression of nr2f2, nr2f1a and s1pr1 had no significant difference from the control. In contrast to in vitro chip analysis, tie2, pik3r2, foxo1b and egfl7 showed consistent high expression in individual mutant group. CONCLUSIONS: TIE2-R849W associated with hereditary venous malformations could induce abnormal development of zebrafish CVP, which implies a possible relationship between VM and egfl7.

Key words: Venous malformations, Caudal vein plexus, Zebrafish, Egfl7

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