内皮细胞特异性表达血管内皮生长因子基因载体的构建及表达特性验证(英文)

Construction of vector specifically expressed vascular endothelial growth factor gene in endothelial cells and research of its expressive character

目的构建在内皮细胞特异性表达人血管内皮生长因子(vascular endothelial growth factor,VEGF)基因的真核表达载体,验证HRE.ppET-1调控元件的内皮细胞表达特异性。方法用聚合酶链反应(PCR)方法直接合成缺氧反应元件(hypoxia-responsive element,HRE),人前内皮素原-1基因(human preproendothe-lin-1,ppET-1)启动子、VEGF元件,通过PCR、酶切、连接等一系列核酸分子操作,将各元件亚克隆至pcD-NA3.1载体上,最终组合而成真核表达载体pcDNA3.1-VEGF+Pa,pcDNA3.1(-CMV)-ppET-1+VEGF+Pa,pcDNA3.1(-CMV)-HRE+ppET-1+VEGF+Pa;脂质体转染离体培养的脐静脉内皮细胞(HUVEC)和肝细胞,荧光显微镜下观察GFP表达,验证HRE.ppET-1调控元件在内皮细胞中的特异性。结果各载体经酶切鉴定和测序证实。GFP的表达显示HRE.ppET-1调控元件在内皮细胞中可有效转录外源基因,在非内皮细胞中表达极弱。结论成功构建能在内皮细胞特异性表达人VEGF基因的真核表达载体;HRE.ppET-1调控元件具有内皮细胞表达特异性,为进一步利用VEGF进行缺血性脑损伤的基因治疗奠定良好基础。

【Objective】To construct the eukaryotic expression vector which specifically expressed vascular endothelial growth factor（VEGF） gene in endothelial cells and to research initially its expression specialty in endothelial cells under the regulation of HRE.ppET-1 element.【Methods】Hypoxia-responsive element（HRE）,human preproendothelin-1（ppET-1） promoter,VEGF element were synthesized by polymerase chain reaction（PCR）.By using a series of nucleate molecule handling methods like PCR,enzyme digestion and conjunction,each element was subcloned to pcDNA3.1 vector to make up eukaryotic expression vectors such as pEGFP-HRE.ppET-1,pcDNA3.1-VEGF＋Pa,pcDNA3.1（-CMV）-ppET-1＋VEGF＋Pa,pcDNA3.1（-CMV）-HRE.ppET-1＋VEGF＋Pa.The recombinant vectors were transfected into HUVEC and HL7702 cells with lipofection method.GFP expressions were observed by fluorescence microscope to validate the specificity in endothelial cells under the regulation of HRE.ppET-1 element.【Results】Each vector was identified by digestion and sequencing analysis.The expressions of GFP revealed that exterior gene was effectively transcripted in endothelial cells under regulation of HRE.ppET-1,while in non endothelial cells the expressions of GFP were very weak.【Conclusions】The eukaryotic expression vectors have been constructed,which specifically express vascular endothelial growth factor（VEGF） gene in endothelial cells.The HRE.ppET-1 element has the expression specialty in endothelial cells,the vector could be a potentially useful tool for vascular targeting gene therapy in ischemic brain damage.