抑制Rac1蛋白活化阻碍胚胎发育早期血管新生  

Inhibition of Rac1 Activity Hinders Vasculogenesis During Early Embryonic Development

作　　者：张剑[1] 韩雅玲[1] 康建[1] 张效林[1] 齐岩梅[1] 闫承慧[1] 李少华 

机构地区：[1]沈阳军区总医院全军心血管病研究所心血管内科 [2]Department of Pathology and Laboratory Medicine,Robert Wood Johnson Medical School,New Jersey 08854,USA

出　　处：《生物化学与生物物理进展》2009年第6期696-701,共6页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(面上课题30770793;30800465)~~

摘　　要：小G蛋白Rac1在胚胎发育早期血管形成尤其是内皮发生过程中的作用尚不清楚.采用胚胎干细胞(ESCs)为模型,建立稳定表达持续表达型Rac1(G12V)和显性失活型Rac1(T17N)编码序列的小鼠ESCs并制备胚胎小体(EBs),诱导分化后观察Rac1(G12V)和Rac1(T17N)对内皮细胞分化和迁移功能的影响.采用相差显微镜观察EBs发育和分化特征,Pull down分析Rac1表达变化,免疫荧光染色和Western blot分析内皮分化标志物,Matrigel凝胶实验观察血管索形成.结果表明,无论过表达或抑制Rac1的活化,并不影响EBs发育,均可形成典型的EBs胚层结构.抑制Rac1活化对内皮细胞系的发育无影响,但分化的内皮细胞不能连接成血管网.活化的Rac1表达减少,细胞迁移受到明显抑制.抑制Rac1活化导致细胞骨架F-actin排布紊乱.以上结果提示,Rac1影响胚胎早期血管发育的因素是抑制细胞游走,后者可能是通过F-actin机制所介导.Whether small G protein Racl plays a role in regulating blood vessel formation especially endothelial cells（ECs） differentiation during early embryonic vascular development remains unclear. Murine embryonic stem cells （ESCs） lines stably expressing the constitutively active Rac 1 mutant （Rac 1G 12V） or dominant negative Rac 1 mutant （Rac 1T 17N） and the resulting embryoid bodies （EBs） were established as models, to observe the effects of Rac 1 on the differentiation and migration of ECs. Using contrast phase microscope to observe the development and differentiation features of EBs; pull-down assay to analyze the expression of Racl activity; immunofluorescence staining and Western blot to inspect the differentiation markers of ECs; matrigel model to observe the assembly of endothelial network. It showed that neither increase nor suppression of Rac1 activity significantly affect the differentiation of EBs to form the typical primitive germ layers. Expression of dominant negative Racl did not affect ECs differentiation, but it significantly inhibited cell migration, furthermore completely blocked the assembly of vessel network. Actin stress fibers were largely absent in Rac1T17N-expressing cells. Expression of Rac1T17N inhibited FAK activity while Rac1G12V did not affect it compared with control group. These results indicated that the effect factor of Rac 1 on the vasculogenesis of EBs was to inhibit cell migration which probably mediated by F-actin mechanism.

关 键 词：胚胎干细胞 RAC1 分化 血管发育 

分 类 号：Q954.4[生物学—动物学] R329.2[医药卫生—人体解剖和组织胚胎学]