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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SP6RNA聚合酶体外合成caPak1mRNA,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30~36h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。
Objective To investigate the effect of p21-activated kinase 1 (Pak1) on the development of embryonic blood vessels and to explore its related mechanism. Methods A recombinant overexpression plasmid PCS2-caPak1, consisting of constitutively active Pak1 (caPak1), was used as a template to synthesize caPak1 mRNA with SP6 RNA polymerase in vitro and then injected into zebrafish single-cell stage embryos by microinjection to observe embryonic development And phenotypic changes, using transmission electron microscopy to further confirm the embryonic vascular ultrastructure. PCS2-caPak1 was transfected into human umbilical vein endothelial cells (HUVEC) by lipofectamine 2000. The viability of HUVECs was detected by MTT assay. The apoptosis of HUVECs was analyzed by flow cytometry. Results The development of zebrafish embryos was observed at 30-36 h after fertilization. The overexpression of Pak1 was found in the zebrafish oocytes, with the proportion of hemorrhage about 25% -30%. The structure of vascular endothelial cells was observed under transmission electron microscope. After the cells became round and partially detached, the cell viability decreased (P <0.01) by MTT assay. The apoptosis of endothelial cells was found by flow cytometry (P <0.01). Conclusion Pak1 overexpression may affect embryonic vascularization by increasing endothelial cell apoptosis.