FLT-1启动子在血管内皮细胞中特异生物活性的检测及其意义

目的:构建带有组织特异性FLT-1启动子的真核表达载体，检测其在转染的人脐静脉内皮细胞（HUVEC）中对荧光素酶报告基因表达的驱动能力。方法：采用PCR扩增FLT-1启动子，插入到pGL3-Basic-luc载体中，构建携带FLT-1启动子的真核表达载体pGL3-FLT-Basic-luc，经脂质体法转染HUVEC、HepG2、NIH3T3和HEK293细胞，于转染48h后采用双荧光报告系统检测荧光素酶表达活性。 结果:酶切及测序证实构建的pGL3-FLT-Basic-luc载体中含有序列正确的FLT-1基因启动子，双荧光报告系统检测显示，转染的HUVEC细胞其荧光素酶活性明显高于HEK293细胞（P<0.01），而转染的HepG2和NIH3T3细胞中未检测出荧光素酶表达。结论:克隆的FLT-1启动子具有较高的血管内皮特异性转录活性，可作为血管疾病靶向基因治疗的启动子来源。图

Identification of tissues specific promoter of FLT-1 and detection its specific activity in transfected HUVEC

Objective:To construct pGL3 Basic eukaryotic expression vector containing tissues specific promoter of FLT-1 and explore the activity of this promoter in HUVEC cells. Methods: The FLT-1 gene promoter was amplified by polymerase chain reaction and cloned into pGL3 Basic vector to construct pGL3 Basic eukaryotic expression vector containing FLT-1 gene promoter(pGL3-FLT-Basic-luc)．The purified pGL3-FLT-Basic-luc was transiently transfected into HUVEC cell and HepG2、NIH3T3、HEK293 cell using liposome transfection reagent, and the activity of luciferase was determined with Dual-Luciferase Reporter Assay System 48h later．Results: DNA sequencing and digestion confirmed that the recombinant of plamid pGL3-FLT-Basic-luc contained FLT-1 promoter sequence.The activity of luciferase in HUVEC was much higher than in HEK293 after transfection of pGL3-Basic-luc, and little activity of luciferase was detected in other two cells．Conclusion: pGL3 Basic eukaryotic expression vector containing tissues specific promoter of FLT-1 was successtully constructed，which might be a potential therapeutic reagent for endothelium-targeted gene therapies for vascular disease.