中文摘要：

为克隆小鼠胎肝激酶-1（fetal liver kinase-1，FLK-1）基因上游启动子序列，并观察其不同截短片段在小鼠血管内皮细胞中的启动子活性，以小鼠全基因组为模板，通过PCR扩增方法获得-258～＋299bp、-96-＋299bp、-71～＋299bp、-36～＋299bp大小的FLK-1启动子片段，将其定向克隆入pGL3．Basic，构建荧光素酶报告基因载体，并制备NF-κB结合位点的突变或缺失体．在阳离子脂质体介导下，报告基因载体瞬时转染小鼠血管内皮细胞株SVEC4．10．结果发现，在小鼠血管内皮细胞中，各FLK-1启动子片段均有活性；-71-36bD区存在FLK-1启动子的核心调控元件．针对该区域NF-κB结合位点进行突变或缺失，能导致启动子活性显著降低；凝胶电泳迁移率实验表明该区段能结合转录因子NF-κB．结果提示，成功克隆了在血管内皮细胞中具有活性的FLK-1上游启动子序列，NF-κB是决定其基本活性的重要转录因子，为进一步研究FLK-1基因的转录调控机制奠定了基础．

英文摘要：

To clone the upstream promoter sequence of mouse fetal liver kinase-1 （FLK-1） and investigate the activities of its truncated segments in mouse vascular endothelial cells, four segments of FLK-1 promoter, -258- ＋299 bp, -96- ＋299 bp, -71- ＋299 bp and -36- ＋299 bp, were obtained by PCR amplification from mouse genomic DNA and were subcloned into pGL3-Basic to construct luciferase reporter vectors. The mutation or deletion of NF-κB binding site within FLK-1 promoter was prepared. The recombinant reporter vectors were transiently transfected into mouse vascular endothelial cell line SVEC4-10 by Lipofectamine 2000. Results showed that all these FLK-1 promoters presented activities in mouse vascular endothelial cells. The region of - 71 - - 36 bp contained the core element of FLK-1 promoter. Mutation or deletion of NF-κB binding site within this region resulted in significant decrease of promoter activity. EMSA demonstrated that this region could bind NF-κB. These results suggest that the upstream promoter sequences of FLK-1 with activities in vascular endothelial ceils, were obtained successfully, in which NF-κB was an important transcription factor determining its basic promoter activity. These results established a basis for further studying transcriptional regulation mechanisms of FLK-1 gene.