向文碧,周栋珍,李志操,周艳华,张鹏,崔冬冰,何志旭,舒莉萍
XIANG Wenbi,ZHOU Dongzhen,LI Zhicao,ZHOU Yanhua,ZHENG Peng,CUI Dongbing,HE Zhixu,SHU Liping

• 1:贵州医科大学细胞工程生物医药技术国家地方联合工程实验室组织工程与干细胞实验中心
• 2:中国医学科学院成体干细胞转化研究重点实验室
• 3:贵州医科大学儿科学教研室
• 4:贵州大学

摘要(Abstract)：

目的:探讨斑马鱼体内髓样特异性过氧化物酶(mpx)在胚胎发育过程中的表达模式。方法:按Trizol法提取斑马鱼总RNA后逆转录成c DNA,再以c DNA为模板RT-PCR扩增mpx基因片段;用基因重组技术构建p CS2~+-mpx重组质粒后将质粒酶切线性化,T3聚合酶制备mpx反义RNA探针,用全胚胎原位杂交检测mpx基因的表达。结果:成功构建了p CS2~+-mpx重组质粒,原位杂交结果显示在24 hpf,mpx基因在斑马鱼胚胎中间细胞团表达;从24 hpf开始,mpx基因在斑马鱼胚胎卵黄囊表面、心脏和轴向脉管系统中高表达,一直持续到72 hpf;尤其是在72 hpf,mpx基因在整个胚胎头部、卵黄囊表面、尾部中弥散表达。结论:mpx基因在斑马鱼心血管系统发育的时间和部位中都有表达,mpx基因可能参与调控斑马鱼心血管系统发育。
Objective: To investigate the expression pattern of myeloid-specific peroxidase( mpx) in embryonic development in zebrafish. Methods: The total RNA of zebrafish was extracted by Trizol method and then reversely transcribed into cDNA,which served as a template to amplify mpx gene fragment by RT-PCR. mpx gene fragment was ligated into pCS2 to obtain the recombinant plasmid pCS2~+-mpx,which was further linearized. Digoxigenin-labeled mpx antisense RNA probe was synthesized in vitro by T3 RNA polymerase. After the embryos of different phases were fixed,the expression of mpx gene in zebrafish embryos was detected by whole embryo in situ hybridization.Results: The pCS2~+-mpx recombinant plasmid was successfully constructed. In situ hybridization showed that the mpx gene was expressed in intermediate cell mass at 24 hpf and highly expressed in the zebrafish embryonic yolk sac surface,heart and axial vasculature from 24 to 72 hpf. Especially,mpx gene is expressed throughout the embryonic head,the surface of the yolk sac,and the tail at 72 hpf.Conclusion: mpx gene expresses at the time and location of zebrafish cardiovascular system development,suggesting that mpx gene may be involved in the regulation of zebrafish cardiovascular system development.

关键词(KeyWords)： 斑马鱼;心血管疾病;早期胚胎发育;mpx基因;全胚胎原位杂交;心血管
zebrafish;cardiovascular diseases(CVD);early embryo development;mpx gene;whole embryo in situ hybridization;cardiovascular

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31860325);; 中国医学科学院中央级公益性科研院所基本科研业务费专项资金资助(2018PT31048);; 贵州省科技厅科技平台及人才团队计划[(2017)5611]

作者(Author): 向文碧,周栋珍,李志操,周艳华,张鹏,崔冬冰,何志旭,舒莉萍
XIANG Wenbi,ZHOU Dongzhen,LI Zhicao,ZHOU Yanhua,ZHENG Peng,CUI Dongbing,HE Zhixu,SHU Liping

DOI: 10.19367/j.cnki.2096-8388.2020.05.005

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