微小染色体维持蛋白5在斑马鱼脑发育中的作用及机制研究  

The Role of Minichromosome Maintenance Protein 5 in Zebrafish Brain Development and It′s Mechanism

作　　者：吴永梅[1,2] 黄四洲 苏炳银[1,2] Wu Yongmei;Huang Sizhou;Su Bingyin(Department of Histology and Embryology, Chengdu Medical College, Chengdu 610500, China;Key Laboratory of Development and Regeneration of Sichuan Province, Chengdu 610500, China)

机构地区：[1]成都医学院组织学与胚胎学教研室,成都610500 [2]发育与再生四川省重点实验室,成都610500

出　　处：《成都医学院学报》2022年第1期1-6,33,共7页Journal of Chengdu Medical College

基　　金：国家自然科学基金(青年科学基金项目)(No:31201092);四川省教育厅基金项目(No:18ZB0161)。

摘　　要：目的观察微小染色体维持蛋白5(mcm5)在斑马鱼脑发育中的作用及机制。方法将90枚斑马鱼胚胎分为3组:对照组[CONT组,注射无意义的吗啉基寡核苷酸(MO)]、mcm5 MO组(注射mcm5特异的MO)和mcm5 MO+mcm5mRNA组(同时注射mcm5特异的MO和mcm5 mRNA),每组30枚,观察mcm5 MO组和CONT组脑部发育情况,并用mcm5 mRNA援救敲降造成的发育缺陷;利用同位素标记相对和绝对定量(iTRAQ)技术分析mcm5 MO组和CONT组之间蛋白质组的变化;采用PH3抗体染色和荧光定量PCR(qPCR)检测斑马鱼脑部增殖细胞的变化以及增殖相关基因的表达量。结果与CONT组相比,mcm5 MO组表现出脑部变小的特征,其中眼睛、中脑和后脑脑室变小较明显。iTRAQ实验结果显示,Hippo信号通路中14-3-3蛋白表达量增加。PH3抗体染色结果表明,mcm5 MO组增殖细胞明显减少,增殖相关基因cycline、cdk2、cdc25b的表达量明显降低。结论mcm5通过Hippo信号通路调节斑马鱼脑部细胞的增殖,影响斑马鱼头部发育。Objective To investigate the role and mechanism of minichromosome maintenance protein 5(mcm5)in zebrafish brain development.Methods Zebrafish embryos were divided into three groups CONT group(n=30,injected with morpholino oligonucleotide that would do nothing),mcm5 MO group(n=30,injected with mcm5 specific morpholino oligonucleotide to knock down mcm5),and mcm5 MO+mcm5 mRNA rescue group(n=30,injected with mcm5 specific morpholino oligonucleotide and mcm5 mRNA at the same time,the developmental defects caused by mcm5 MO were rescued with mcm5 mRNA).The brain development of mcm5 MO group and CONT group was observed under a microscope.The iTRAQ technology was used to analyze the proteomic change difference between the mcm5 MO group and the CONT group.Finally,PH3 antibody staining and quantitative polymerase chain reaction(qPCR)were used to detect the changes of zebrafish brain proliferation cells and the expression of proliferation-related genes.Results Compared with the CONT group,the mcm5 MO group showed reduced brain size,especially in the eyes,midbrain and hindbrain ventricles.The results of the iTRAQ experiment showed that the expression of 14-3-3 proteins of Hippo signaling pathway was increased.At the same time,PH3 antibody staining results showed that the proliferation cells were significantly reduced in the mcm5 MO group,and the expression of proliferation-related genes cycline,cdk2,and cdc25b was significantly reduced.Conclusion mcm5 regulates the proliferation of zebrafish brain cells through Hippo signaling pathway,leading to smaller zebrafish head.

关 键 词：微小染色体维持蛋白5 脑发育 Hippo信号通路 增殖 斑马鱼 

分 类 号：R742.8[医药卫生—神经病学与精神病学]