dusp1敲降对低温诱导斑马鱼ZF4细胞凋亡的作用
作者:
作者单位:

上海海洋大学 水产与生命学院, 上海海洋大学省部共建水产种质资源发掘与利用教育部重点实验室, 上海 201306

作者简介:

牛虹博(1989-),女,硕士,专业方向为水产养殖.E-mail:1039428340@qq.com

中图分类号:

S917

基金项目:

国家基金委重点项目(31130049);国家自然科学基金面上项目(31572611);上海高校水产高峰学科建设项目.


The role of dusp1 downregulation in apoptosis of zebrafish ZF4 cells under cold stress
Author:
Affiliation:

College of Fisheries and Life Sciences, Shanghai Ocean University;Key laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China

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    摘要:

    为了研究双特异性磷酸酶(dual-specificity phosphatase 1,dusp1-shRNA1,plko.1--shRNA2,plko.1-negative control(nc),plko.1-EGFP的重组质粒,并分别与慢病毒包装质粒pCMV-DR8.9.1、pCMV-VSVG共同转染至293T细胞中,经293T细胞包装的病毒,感染斑马鱼()胚胎成纤维样细胞(zebrafish embryos fibroblast like cell line,ZF4),RT-PCR检测表明成功被敲降。经嘌呤霉素筛选获得稳定表达细胞系,将细胞于28℃培养(正常对照组)或经10℃低温处理10 d,使用annexin V-FITC/Propidium lodide(PI)双色标记流式细胞术检测ZF4细胞凋亡情况。结果显示:低温胁迫下-shRNA2敲降,-kd1活细胞数显著低于nc组(参与鱼类冷应激过程,并在低温情况下保护细胞。该研究为后期对斑马鱼细胞低温胁迫实验奠定了基础,其中在细胞凋亡信号通路中起到重要调控作用,为低温下研究斑马鱼基因的分子机制提供新的思路。

    Abstract:

    The purpose of this study was to observe whether dual-specificity phosphatase 1 () cells under cold stress. The recombinant plasmids plko.1--shRNA2, plko.1-negative control, and plko.1-EGFP were constructed using the restriction enzymes . These plasmids were separately transduced into 293T cells together with the lentiviral packaging vectors pCMV-DR8.9.1 and pCMV-VSVG. The virus packaged by 293T cells was used to infect the ZF4 cell line derived from zebrafish, and analysis by real-time polymerase chain reaction showed that knockdown ZF4 cell line, the cells were cultured at 28℃ (normal temperature control) or 10℃ (cold stress) for 10 days. Afterwards, they were stained with propidium iodide and annexin V-fluorescein isothiocyanate, then analyzed by flow cytometry. The results showed that the rate of apoptosis of the -knockdown cells increased significantly more under low temperature than that of the wild-type control cells did, as compared with the normal temperature control group. Furthermore, we designed two small hairpin RNAs named kd1 and kd2 based on the coding sequence of . Under cold stress, the frequencies of late and early apoptotic cells in the kd1-treated group were significantly higher, and the frequency of living cells was significantly lower, than those of the negative control group (<0.05). The cells cultured at 28℃ showed no significant difference in the rate of apoptosis according to silencing. However, under cold stress, apoptosis was significantly greater in the is involved in several physiological processes of fish, and it negatively regulates MAPK family proteins; for example, it inhibits p38 phosphorylation. This paper provides a new insight into the regulation of the apoptotic response of fish cells under low-temperature conditions by expression may relieve the inhibition of the MAPK p38 signaling pathway, which promotes ZF4 cell apoptosis under cold stress.

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牛虹博,胡鹏,程鹏丽,储旭,胡春兰,陈良标. dusp1敲降对低温诱导斑马鱼ZF4细胞凋亡的作用[J].中国水产科学,2017,24(5):995-1002
NIU Hongbo, HU Peng, CHENG Pengli, CHU Xu, HU Chunlan, CHEN Liangbiao. The role of dusp1 downregulation in apoptosis of zebrafish ZF4 cells under cold stress[J]. Journal of Fishery Sciences of China,2017,24(5):995-1002

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  • 在线发布日期: 2017-09-12
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