敲降组蛋白去乙酰化酶HDAC8对斑马鱼ZF4细胞存活率的影响  

Effect of Knockdown of Histone Deacetylase HDAC8 on the Survival Rate of Danio rerio ZF4 Cells

作　　者：李飞[1] 罗军涛[1] 燕晓霞 陈静 张婷婷[1] 韩兵社[1] 张俊芳[1] Li Fei;Luo Juntao;Yan Xiaoxia;Chen Jing;Zhang Tingting;Han Bingshe;Zhang Junfang(Key Lab of Ministry of Education for Exploration and Utilization of Aquatic Genetic Resources,International Research Center for Marine Biosciences,National Demonstration Center for Experimental Fisheries Science Education,College of Fisheries and Life Science,Shanghai Ocean University,Shanghai,201306)

机构地区：[1]上海海洋大学水产与生命科学学院,水产科学国家级实验教学示范中心,海洋生物科学国际联合研究中心,水产种质资源发掘与利用教育部重点实验室,上海201306

出　　处：《基因组学与应用生物学》2021年第3期985-990,共6页Genomics and Applied Biology

基　　金：上海市科技兴农重点攻关项目(沪农科创字〔2019〕第1-2号);国家自然科学基金面上项目(81770165)共同资助。

摘　　要：组蛋白去乙酰化酶(HDACs)作为表观遗传酶类的重要一员,能够通过去乙酰化核内组蛋白的方式在细胞转录调控中发挥重要作用,然而,其在鱼类生理中的作用目前报道较少。本研究设计合成了斑马鱼HDAC8的shRNA,用EcoR I和AgeI酶切构建了pLKO.1-hdac8-shRNA重组质粒,同时构建了一个阴性对照重组质粒(nc),接着将pCMV-VSVG、pCMV-Dr8.91两个慢病毒包装质粒分别与设计好的重组质粒共转染到HEK-293T细胞中进行病毒包装。收集病毒并用其感染ZF4细胞,RT-qPCR检测表明ZF4细胞中hdac8成功被敲降。随后使用台盼蓝染色法检测ZF4细胞存活率,并进一步检测了细胞内凋亡相关基因bax、bcl2、caspase-3的表达情况。研究发现:与对照组(nc)细胞相比,敲降hdac8的细胞存活率显著下降(P<0.05),bax及caspase-3的表达量在敲降hdac8的细胞内显著上调,意味着敲降hdac8的细胞死亡的增加可能是通过上调凋亡水平引起的。本研究为后期对斑马鱼在生长发育过程中及环境压力下的凋亡机制的研究提供基础。Histone deacetylases(HDACs),important members of the epigenetic enzymes,play an important role in cellular transcriptional regulation through deacetylation of nuclear histones.However,their roles in fish physiology have been little explored.This study was designed to synthesize shRNA of zebrfish HDAC8,construct pLKO.1-hdac8-shRNA recombinant plasmid by EcoRⅠand AgeⅠenzyme digestion,and a negative control recombinant plasmid(nc)at the same time.Then,two lentivirus packaging plasmids,pCMV-VSVG and pCMV-Dr8.91,were co-transfected into HEK-293T cells for virus packaging,respectively.RT-qPCR detection showed that hdac8 was successfully knocked down.The survival rate of ZF4 cells was detected by trypan blue staining,and the expression of apoptosis-related genes bax,bcl2 and caspase-3 were further detected.The results showed that the survival rate of hdac8 knockdown cells was significantly lower than that of nc cells(P<0.05),and the expression of apoptosisrelated genes bax and caspase-3 in hdac8 knockdown cells was significantly up-regulated,suggesting that the increased cell mortality by knocking down hdac8 was caused by up-regulation of apoptosis.This study provided a basis for further study on the mechanism of zebrafish HDAC8 during growth and development process and under the environmental pressure.

关 键 词：HDAC8 斑马鱼ZF4细胞 细胞存活率 

分 类 号：S917.4[农业科学—水产科学]