首页  期刊介绍 征稿简则 编委会 期刊征订 广告服务 留言板 联系我们 English

中国应用生理学杂志 ›› 2019, Vol. 35 ›› Issue (1): 5-8.doi: 10.12047/j.cjap.5709.2019.002

• 研究论文 • 上一篇    下一篇

EGR1基因在牛骨骼肌卫星细胞分化不同时期的表达及定位

张伟伟1,2,邵淑丽1,2△,潘阳1,李珊珊1   

  1. 1. 齐齐哈尔大学生命科学与农林学院,
    2. 抗性基因工程与寒地生物多样性保护黑龙江省重点实验室, 黑龙江 齐齐哈尔 161006
  • 收稿日期:2018-05-30 出版日期:2019-01-28 发布日期:2019-06-27
  • 通讯作者: Tel: 0452-2738219; E-mail: shshl32@163.com
  • 基金资助:
    黑龙江省自然科学基金(C2016057, C201241, C2017068); 黑龙江省省属高等学校基本科研业务费科研项目(135209260, 135109104,135109309); 齐齐哈尔市科学技术计划项目(NYGG-201515, NYGG-201616)

Expression of EGR1 gene and location of EGR1 protein in differentiation of bovine skeletal muscle-derived satellite cells

ZHANG Wei-wei1,2, SHAO Shu-li1,2△, PAN Yang1,2, LI Shan-shan1,2   

  1. 1. College of Life Sciences, Agriculture and Forestry;
    2. Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas; Qiqihar University, Qiqihar 161006
  • Received:2018-05-30 Online:2019-01-28 Published:2019-06-27

摘要: 目的:研究EGR1基因在牛骨骼肌卫星细胞(MDSCs)分化过程的表达、定位及入核机制。方法:以牛的MDSCs为实验材料,在分化培养基中分别分化培养1 d、3 d和5 d,每组3个重复,检测不同分化时间的MDSCs中EGR1基因的表达和EGR1蛋白的定位情况;采用 CRISPRi方法干扰内源EGR1的表达,结合定点突变和激光共聚焦方法初步探索了EGR1蛋白入核的机制。结果:qRT-PCR和Western blot检测结果显示随着分化时间的进行,EGR1 基因在转录水平和蛋白水平的表达都显著高于未分化的细胞,并随时间的延长而表达逐渐升高,分化第3日时表达量最高,随后开始下降。免疫荧光检测到EGR1蛋白主要在分化的MDSCs中表达,并随肌管数量增多而表达量增加。共聚焦结果显示随着细胞分化的进行,部分EGR1蛋白转移进入细胞核。定点突变EGR1蛋白S533A后,分化的MDSCs细胞核内没有检测到EGR1蛋白。结论:在牛骨骼肌卫星细胞分化过程中,EGR1基因转录表达水平升高,部分EGR1蛋白转移入细胞核,且EGR1蛋白C端第533位丝氨酸磷酸化是入核所必需的。

关键词: 牛骨骼肌卫星细胞, 分化, EGR1基因, 入核作用

Abstract: Objective:To investigate the expression of EGR1 gene and the localization of EGR1 protein in bovine skeletal muscle-derived satellite cells (MDSCs), as well as to investigate the mechanism that EGR1 protein enters the nucleus. Methods: Bovine MDSCs were cultured in differentiation medium for 1 day, 3 days and 5 days, respectively, and each group was triplicate. The expression of EGR1 gene and the localization of EGR1 protein were studied at different differentiation period in MDSCs by qRT-PC and Western blot. Moreover, the changes on the expression of endogenous EGR1 gene and EGR1 proteins were explored by CRISPRi, site-directed mutagenesis and laser confocal method. Results: The results from the qRT-PCR and Western blot showed that the expressions of EGR1 gene on transcription level and translation level were significantly higher in differentiated cells than those in undifferentiated cells. The highest expression was found on the third day after the differentiation, and then began to decline. Immunofluorescence assays showed that EGR1 proteins were preferentially expressed in differentiated MDSCs, and increased along with the increase of number of myotubes. Confocal observation revealed that some EGR1 proteins were transferred into the nucleus in the differentiation of cells, however, the EGR1 proteins would not be detected in the differentiated MDSCs nuclei if a site directed mutagenesis (serine533) on EGR1 protein occurred. Conclusion: During the differentiation of bovine skeletal muscle satellite cells, the transcriptional level of EGR1 gene is increased, and some EGR1 proteins are transferred into the nucleus. The serine phosphorylation at position 533 of the C terminal of EGR1 protein is necessary for the nucleus transfer.

Key words: bovine skeletal muscle derived satellite cells, differentiation, EGR1 gene, enter the nucleus

版权所有 © 2015 《中国应用生理学杂志》编辑部
京ICP备16058274号-1
地址:天津市和平区大理道1号,邮编:300050  电话:022-23909086  E-mail:editor@cjap.ac.cn
本系统由北京玛格泰克科技发展有限公司设计开发 技术支持:support@magtech.com.cn