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当前位置: 首页 > 文章 > 低温胁迫诱导斑马鱼ZF4细胞ROS及MAPK相关蛋白表达的影响 中国水产科学 2016,23 (4) 771-776
Position: Home > Articles > Effects of cold stress on ROS production and expression of MAPK proteins in zebrafish ZF4 cells Journal of Fishery Sciences of China 2016,23 (4) 771-776

低温胁迫诱导斑马鱼ZF4细胞ROS及MAPK相关蛋白表达的影响

作  者：
许琼琼;韩兵社;罗军涛;李艳;侯艳雯;胡鹏;张俊芳
单  位：
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海洋大学水产与生命学院
关键词：
ROS;斑马鱼ZF4细胞;低温胁迫;MAPKs;γH2A.X
摘  要：
为了研究鱼类低温下分子调控机制及其与活性氧(reactive oxygen species,ROS)之间的关系,本实验对斑马鱼(Danio rerio)胚胎成纤维ZF4细胞进行不同程度的低温胁迫(18℃和10℃),监测其在不同低温胁迫时间下(1 d、3 d和5 d)ROS的变化以及丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)通路中蛋白的表达情况。结果显示:(1)DCFH-DA探针法表明在低温胁迫作用下,细胞内ROS含量增加。细胞内ROS上升水平与外界胁迫压力成正相关。低温处理3 d后,18℃和10℃的细胞,其ROS含量与对照组(28℃)相比分别显著升高到(1.23±0.04)倍(P<0.05)和(2.31±0.08)倍(P<0.05)。(2)Western Blot检测磷酸化的p38(p-p38)和磷酸化的JNK(p-JNK p54和p-JNK p46)的水平变化,结果显示低温胁迫可以使p38和JNK的活性增强,且均在10℃处理3 d达到最高水平。(3)进一步检测DNA断裂标记蛋白γH2A.X的表达水平,结果显示,无论在18℃还是10℃,其在第3天呈现高表达。本实验初步证实低温胁迫能够诱导斑马鱼细胞ROS的产生;通过对不同时间点的蛋白表达水平检测,发现p-JNK、p-p38和γH2A.X激活时间点与低温诱导ROS表达时间点相吻合。该研究为后期对斑马鱼细胞低温胁迫实验奠定基础,其中低温下处理3 d可以作为一个检测各个蛋白变化的关键时间点。
译  名：
Effects of cold stress on ROS production and expression of MAPK proteins in zebrafish ZF4 cells
作  者：
XU Qiongqiong;HAN Bingshe;LUO Juntao;LI Yan;HOU Yanwen;HU Peng;ZHANG Junfang;Ministry of Education;College of Fisheries and Life Science, Shanghai Ocean University;
关键词：
ROS;;zebrafish ZF4 cells;;low temperature stress;;MAPKs;;γH2A.X
摘  要：
In our previous study, we compared the transcriptome between Antarctic fish and temperate fish and found that genes were involved in reactive oxygen species(ROS) scavenge and Mitogen-activated protein kinase(MAPK) pathway associated with the cold adaptation in fish. To further study the regulatory network and the reactive oxygen species(ROS) homeostasis under cold stress in fish, we examined the level of ROS and the expression of stress-related proteins in zebrafish Danio rerio-derived ZF4 cells exposed to mildly and severely cold temperatures, 18℃ and 10℃, at various time points(1 d, 3 d, and 5 d). Results showed that(1) DCFH-DA probe method was used to determine the level of ROS in cells. We found that the level of ROS in cell positively correlated with the intensity of cold stress. After 3 days of cold treatment, the level of ROS in cells significantly increased to 1.23±0.04(P<0.05) and 2.31±0.08(P<0.05) times, respectively, at 18℃ and 10℃, compared with that in cells at 28℃.(2) Western blot showed that the expression of MAPKs(p-JNK p54, p-JNK p46 and p-p38) was induced under cold stress, and reached the highest at 3 d of 10℃.(3) In addition, the expression of γH2A.X reached peak after 3 days both at 18℃ and 10℃. These findings showed that cold temperature could induce the production of ROS in ZF4 cells. The level of ROS in ZF4 cells is dependent on the stress intensity and duration. We observed the markedly induction of ROS in the cells at the first 3 days of cold treatment, while the expression of p-JNK, p-p38, and γH2A.X was markedly induced at the 3 d, indicating that 3 d of cold treatment might be a key time point to determine the protein expression in ZF4 cells.

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