摘要
背景:血管内皮生长因子和骨形态发生蛋白具有协同促进血管生成的作用。目的:构建在真核细胞中表达人血管内皮生长因子165的红色荧光蛋白表达载体和携带人骨形态发生蛋白2的绿色荧光蛋白表达载体,共转染真核细胞以研究血管内皮生长因子165和骨形态发生蛋白2在293-T细胞内的表达和定位。设计:随机对照实验。单位:解放军第三军医大学大坪医院野战外科研究所国家重点实验室。材料:实验于2002-09/2004-03在解放军第三军医大学大坪医院野战外科研究所国家重点实验室完成。pCDNA3.1(+)/骨形态发生蛋白2由美国UCLA大学Dr.Bostrom惠赠;pDsRed1-N1由ProfessorRogerY.Tsien,UniversityofCalifornia,SanDiego,USA惠赠。pUC18/血管内皮细胞生长因子165,293-Tcells由本实验室保存。方法:根据已知的人血管内皮生长因子165序列,设计在目的片段两端分别携带酶切位点的两条引物,用聚合酶链反应从质粒pUC18/血管内皮生长因子165中扩增出去除终止密码子的人血管内皮生长因子165片段,定向克隆至含报告基因的质粒pDsRed1-N1中,构建重组质粒pDsVEGF165Red1-N1;同时,构建pIRES2-骨形态发生蛋白2-加强型绿色荧光蛋白表达载体。以DOTAP为介导,将重组质粒共转染293-T细胞,48h后用激光共聚焦显微镜检测报告基因红色荧光蛋白和加强型绿色荧光蛋白的表达,使用反转录聚合酶链反应及免疫印迹方法检测血管内皮生长因子165和骨形态发生蛋白2在细胞内表达。主要观察指标:质粒的酶切鉴定及重组质粒在293-T细胞中mRNA及蛋白水平的表达。结果:重组质粒经酶切、聚合酶链反应和DNA序列测定证明构建正确,目的基因在293-T细胞中在mRNA及蛋白水平获得表达。激光共聚焦显微镜观察到红色荧光蛋白和加强型绿色荧光蛋白在细胞内共定位的情况。结论:成功构建pDsVEGF165Red1-N1和pIRES2-骨形态发生蛋白2-加强型绿色荧光蛋白
BACKGROUND： It has been proved that vascular endothelial growth factor 165（VEGFI6s） and bone morphogenetic protein 2（BMP2） can accelerate the vascularization synergistically.
OBJECTIVE：To construct the vectors, pDsVEGF165Red1-N1 and pIRES2- BMPr-enhanced green fluorescent protein（EGFP） followed by co-transfected into HEK 293-T cells,and study their expression and location of VEGF165 and BMP2 in the cells.
DESIGN： A randomized and controlled experiment.
SETTING： National Key Laboratory,Institute of Surgery Research,Daping Hospital,Third Military Medical University. MATERIALS： The experiment was conducted at National Key Laboratory, Institute of Surgery Researeh,Daping Hospital,Third Military Medical University from September 2002 to March 2004. pcDNA3.1/BMP2 （ gift of Dr. Bostrom, UCLA School of Mediine, Los Angeles,USA）.pDsRedl-N 1 （gift of Pro.Roger Y.Tisen,University of California,San Diego,USA）. pUC18/ VEGF165,293-T cells（preserved by our Laboratory）.
METIIODS： According to the nucleotide sequence of hVEGF165, the primers were designed.The hVEGF165 gene without stop codon was amplified by polymerase chain reaction （PCR）.The fragment digested was cloned into the expression vector pDsRed1-N1.Meantime,the plRES2-BMP2-EGFP expression vector was constructed.The two plasmids were co-transfected into HEK 293-T cells.The co-expression and distribution of the VEGF165 and BMP2 were observed with confocal laser microscopy （CLSM） and detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western Blotting.
MAIN OUTCOME MEASURES： Identification of the recombinant plasmids and the expressing mRNA and protein in 293-T cells. RESULTS： The recombinant plasmids were verified correct construction by restriction enzyme analysis, PCR and sequencing. The two genes which were co-transfected could express in HEK 293-T cells.The co-expression of the report genes,RFP and EGFP, were found over the cytoplasm and in the nuclei by CLSM. CONCLUSION： Two report
出处
《中国临床康复》
CSCD
北大核心
2006年第21期174-176,F0003,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家高科技研究发展计划("八六三"计划)(2002AA326020)~~
