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  • 董斐斐,邹俊,荆清,秦永文.Pak1对胚胎血管发育的作用及初步机制[J].第二军医大学学报,2014,35(4):394-398    [点击复制]
  • DONG Fei-fei,ZOU Jun,JING Qing,QIN Yong-wen.Effect of Pak1 on embryonic vascular development and the related mechanisms[J].Acad J Sec Mil Med Univ,2014,35(4):394-398   [点击复制]
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Pak1对胚胎血管发育的作用及初步机制
董斐斐,邹俊,荆清,秦永文*
0
(第二军医大学长海医院心血管内科, 上海 200433
*通信作者)
摘要:
目的 研究p21活化激酶1(p21-activated kinase 1,Pak1)对胚胎血管发育的影响,并初步探讨其相关作用机制。方法 以组成激活型Pak1(constitutively active Pak1,caPak1)的重组过表达质粒PCS2-caPak1为模板,用SP6 RNA聚合酶体外合成caPak1 mRNA,用显微注射法将其注入斑马鱼单细胞期胚胎,观察胚胎发育和表型变化,用透射电镜进一步确认胚胎血管的超微结构。用脂质体转染法将PCS2-caPak1转入人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),用MTT法检测细胞活力,并用流式细胞术分析HUVEC凋亡情况。结果 斑马鱼胚胎发育至受精后30~36 h时,可见过表达组斑马鱼出现头部出血表型,出血比例约25%~30%,透射电镜下可见血管内皮细胞结构破坏;细胞水平过表达Pak1后,细胞变圆并出现部分脱落,MTT检测发现细胞存活减少(P<0.01),流式细胞术分析发现内皮细胞凋亡增加(P<0.01)。结论 过表达Pak1可能通过增加内皮细胞凋亡影响胚胎血管发育。
关键词:  p21活化激酶类  内皮细胞  细胞凋亡  血管发育  斑马鱼
DOI:10.3724/SP.J.1008.2014.00394
投稿时间:2014-03-18修订日期:2014-03-26
基金项目:国家自然科学基金青年项目(31101037).
Effect of Pak1 on embryonic vascular development and the related mechanisms
DONG Fei-fei,ZOU Jun,JING Qing,QIN Yong-wen
(Department of Cardiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
*Corresponding author.)
Abstract:
Objective To investigate the effects of p21-activated kinase 1 (Pak1) on embryonic vascular development and to discuss the underlying mechanisms. Methods In vitro transcription reactions were performed with the linearized PCS2-caPak1 templates and SP6 RNA polymerase. Overexpression of constitutively active Pak1 (caPak1) mRNA in one-cell stage embryos of zebrafish was achieved by microinjection. The morphological changes of the embryos were observed under an inverted microscope and the ultrastructure of vessel was confirmed by transmission electron microscope. PCS2-caPak1 was used to transfect into human umbilical vein endothelial cells (HUVECs) via Lipofectamine LTX; cell viability was tested by MTT based assay; and apoptosis of HUVECs was analyzed by flow cytometry with Annexin Ⅴ/7-AAD double-staining. Results Obvious cerebral hemorrhage was observed in the embryo of zebrafish at 30-36 h after fertilization, and the hemorrhage phenotype was observed in the caPak1 overexpression group, with the incidence of hemorrhage being 25%-30%. In the caPak1 overexpression group, transmission electron microscope found damaged vascular endothelial cells in the bleeding sites. Inverted microscope also showed abnormal morphology of cultured cells with caPak1 overexpression, rounded up and started to detach from the dishes. MTT assay showed significantly decreased cell survival in the caPak1 overexpression group (P<0.01), and flow cytometry demonstrated a significantly higher rate of cell apoptosis (P<0.01). Conclusion Pak1 overexpression may affect embryonic vasculogenesis through inducing apoptosis of vascular endothelial cells in zebrafish.
Key words:  p21-activated kinases  endothelial cells  apoptosis  vascular development  zebrafish