PGC-1α敲降对斑马鱼细胞冷应激过程的影响

Effect of PGC-1α Knockdown on Zebrafish Cells under Cold Stress

过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ,PPARγ)辅助激活因子1α(PPARγcoactivator-1α,PGC-1α)作为一个重要的转录激活因子,参与多种生理活动的调节。本研究探究了PGC-1α在斑马鱼(Danio rerio)ZF4细胞(胚胎发育3 d成纤维样细胞)冷应激过程中所起的作用。通过慢病毒质粒转染干扰PGC-1α基因的表达,并用嘌呤霉素筛选得到稳转细胞系,给与其18℃7 d和10℃7 d的低温刺激后使用CM-H2DCFDA探针染色,通过流式细胞仪检测细胞内活性氧(reactive oxygen species,ROS)含量;JC-1探针染色,流式细胞仪检测低温处理后PGC-1α敲降的细胞线粒体膜电位的变化。研究表明,在低温刺激下,PGC-1α基因未敲降的ZF4细胞内ROS含量升高,线粒体膜电位下降;而在PGC-1α基因敲降的ZF4细胞内,ROS含量在前者的水平上进一步升高,线粒体膜电位的下降也加剧,故PGC-1α在ZF4细胞冷应激过程中对细胞起到保护作用,能维持细胞内ROS含量和线粒体膜电位水平。本研究对PGC-1α基因在斑马鱼ZF4细胞冷应激过程中所起的功能进行初步探究,为鱼类在低温下的适应性研究提供依据。

PGC-1αinvolves in the regulation of various physiological activities as an important transcriptional activator.In order to investigate the role of PGC-1αunder cold stress of ZF4 cells(zebrafish embryonic development 3 days fibroblast-like cells),we suppressed the expression of PGC-1αusing lentiviral plasmids,then treated the cells with puromycin to select the stable cell lines.After that the cells were under cold treatment at 18℃for 7 days or 10℃for 7 days,and stained with CM-H2DCFDA probe to detect the ROS content in cells by flow cytometry.In addition,the cells were stained with JC-1 probe to detect the mitochondrial membrane potential changes of PGC-1αby flow cytometry after cold treatment.The research results showed that under cold stimulation,ROS in ZF4 cells without PGC-1αknockdown was increased,and mitochondrial membrane potential decreased,whereas in ZF4 cells which PGC-1αwas knockdown,ROS increased,comparing to the former level.Besides,mitochondrial membrane potential was decreased rather than the negative control group.These results indicated that PGC-1αof ZF4 cells could protect cells from cold stress,and could maintain intracellular ROS content and mitochondrial membrane potential.This study preliminary explored the function of PGC-1αon ZF4 cells during cold acclimation, and provided a basis for adaption of fish under cold stress.