EDU脉冲追踪小鼠心脏第二心场细胞迁移方法的优化

doi:10.13560/j.cnki.biotech.bull.1985.2018-0518

生物技术通报 ›› 2018, Vol. 34 ›› Issue (12): 84-89.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0518

EDU脉冲追踪小鼠心脏第二心场细胞迁移方法的优化

袁白银, 刘钟颖

武汉科技大学生命科学与健康学院,武汉 430065

收稿日期:2018-06-05 出版日期:2018-12-26 发布日期:2018-12-24
作者简介:袁白银,女,博士,研究方向：生物学;E-mail：yuanby@wust.edu.cn
基金资助:
国家自然科学基金项目（31701266）,湖北省自然科学基金项目（2016CFB172）

Optimization of EDU Pulse Method for Tracing the Migration of Mouse Second Heart Field Cells

YUAN Bai-yin, LIU Zhong-ying

College of Life Science and Health,Wuhan University of Science and Technology,Wuhan 430065

Received:2018-06-05 Published:2018-12-26 Online:2018-12-24

摘要/Abstract

摘要： 流出道（Outflow tract,OFT）发育畸形约占出生时检测到的人类先天性心脏病的 30%,OFT 和右室是由第二心场（Second heart field,SHF）祖细胞发育形成。SHF 祖细胞向心管部署、迁移使心管得以足够的伸展,是心脏形态发生所必需的。然而,迄今 SHF 发育及迁移部署过程仍不清楚。建立标记、追踪小鼠心脏第二心场细胞迁移的方法,对于阐明哺乳动物心脏 SHF 迁移、部署的细胞生物学过程具有重要意义。首先采用文献报道的 EDU（5-乙炔基-2’脱氧尿嘧啶核苷）脉冲追踪方法及剂量（10 mg/kg）,发现大部分胚胎并不能被 EDU 标记。接下来,分别对 EDU 标记的剂量以及脉冲标记的时间进行优化,分析 EDU 在 SHF 细胞中的标记情况、EDU 标记的 SHF 细胞向 OFT 的迁移情况。发现 EDU 剂量增加到 40 mg/kg 能对胚胎进行有效的标记（由 6 只孕鼠获得的 31 只胚胎均被有效的标记）,且不会对孕鼠和胚胎造成毒性;EDU 脉冲标记 2 h 和 4 h,EDU 在 SHF 细胞中的标记效率、EDU 标记 SHF 细胞迁移进入远端 OFT 的距离均没有差异。研究表明使用 40 mg/kg 的 EDU 脉冲剂量和 2 h 的标记时间,可对胚胎的 SHF 细胞进行有效的标记,经过 19 h 的追踪,EDU 标记的细胞能够迁移进入到远端 OFT 中。

关键词: 第二心场细胞, EDU, 细胞迁移, 小鼠

Abstract: Outflow tract（OFT）malformation accounts for about 30% of detected human congenital heart diseases at birth. OFT and right ventricle are developed from second heart field（SHF）. Deployment and migration of SHF cells to heart tube is essential for sufficient lengthening of heart tube and subsequent heart morphogenesis. However,the development,migration and deployment process of SHF is still unclear. Establishing a method of marking and tracing the migration of second heart field cells in the mouse heart is of great significance in elucidating the cellular biological processes of SHF migration and deployment in mammalian hearts. Firstly,we adapted EDU（5-ethynyl-2'-deoxyuridine）pulse-chase assay with dose（10 mg/kg）reported in the literature,and the most embryo were not labeled by EDU. Following,we optimized the dosage and pulse time by analyzing the labeling effects of EDU in SHF cells and the migration of EDU-labeled SHF cells to OFT. The dosage of 40 mg/kg demonstrated effective labeling on SHF cells（31 embryos from 6 pregnant mice were efficiently labeled）,and no toxicity to embryos and pregnant mice. Moreover,2 h- and 4 h- EDU pulse period showed no difference in EDU labeling efficiency and the migratory distance to the distal OFT. Taking above results together,the 40 mg/kg EDU pulse dose and 2 h EDU pulse period presented effective EDU labeling of SHF cells in embryos,and many EDU labeling SHF cells can be detected in the distal OFT after 19 h tracing period.

Key words: second heart field cells, EDU, cell migration, mouse

袁白银, 刘钟颖. EDU脉冲追踪小鼠心脏第二心场细胞迁移方法的优化[J]. 生物技术通报, 2018, 34(12): 84-89.

YUAN Bai-yin, LIU Zhong-ying. Optimization of EDU Pulse Method for Tracing the Migration of Mouse Second Heart Field Cells[J]. Biotechnology Bulletin, 2018, 34(12): 84-89.

参考文献

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EDU脉冲追踪小鼠心脏第二心场细胞迁移方法的优化

Optimization of EDU Pulse Method for Tracing the Migration of Mouse Second Heart Field Cells

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