真核表达载体pcDNA3.1/VEGF-B-EGFP的构建及在骨髓间质干细胞中的表达  

Construction of eukaryotic expression plasmid of pcDNA3.1/VEGF-B-EGFP and its expression in bone marrow mesenchymal stem cells

作　　者：张勇[1] 王胜[2] 王惠[3] 

机构地区：[1]济南军区总医院心外科,济南市250031 [2]山东省军区门诊部,济南市250013 [3]济南军区总医院超声诊断科,济南市250031

出　　处：《中华实用诊断与治疗杂志》2012年第4期364-367,共4页Journal of Chinese Practical Diagnosis and Therapy

摘　　要：目的探讨血管内皮细胞生长因子B(vascular endothelial cell growth factor-B,VEGF-B)转染人骨髓间质干细胞(marrow mesenchymal stem cells,MSCs)的表达情况。方法将VEGF-B及编码增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的互补脱氧核糖核酸(complementary deoxyribonucleic acid,cDNA)序列通过扩增、酶切、插入pcDNA 3.1质粒的多克隆位点,构建pcDNA 3.1/VEGF-B-EGFP真核表达质粒,应用脂质体介导的基因转染技术,将基因导入MSCs(pcDNA 3.1/VEGF-B-EGFP转染组);应用荧光显微镜观察EGFP的表达情况;流式细胞仪检测转染效率;逆转录-聚合酶链反应、蛋白印迹法检测VEGF-B的表达情况。结果 pcDNA 3.1/VEGF-B-EGFP转染组重组质粒转染MSCs 24h后,荧光显微镜下可见强绿色荧光,流式细胞仪检测转染效率约为15%;RT-PCR检测结果显示,MSCs出现590bp目的基因;Western blot检测显示,在转染后MSCs中有VEGF-B表达。结论构建的VEGF-B真核表达质粒成功转染MSCs,为联合应用MSCs细胞移植和VEGF-B基因治疗心肌缺血的研究奠定了基础。Objective To detect the expression of vascular endothelial cell growth factor（VEGF）-B in human bone marrow mesenchymal stem cells（MSCs）.Methods The recombinant plasmids of pcDNA 3.1/VEGF-B-EGFP were obtained by polymerase chain reaction（PCR） amplification,splicing,and insertion of complementary deoxyribonucleic acid（cDNA） fragments of VEGF-B and EGFP.The target gene was transferred to the third cultured MSCs（pcDNA 3.1/VEGF-B-EGFP transferred group） by using of cationic liposome mediated gene transfection technique.EGFP expression was detected with fluorescence microscopy,and the transfection rate was determined with flow cytometry.Reverse transcriptase PCR（RT-PCR）,Western blot and other techniques were used to detect the expression of VEGF-B gene.Results In pcDNA 3.1/VEGF-B-EGFP transferred group,bright green fluorescence was observed with fluorescence microscopy 24 hours after transfection in MSCs,the rate of transfection analyzed with flow cytometry was 15%.RT-PCR results showed that the genome of VEGF-B transferred MSCs contained a 590bp specific fragment of VEGF-B gene.Western blot results proved VEGF-B protein expression in MSCs transferred by VEGF-B.Conclusion The generation of eukaryotic expression plasmid carrying VEGF-B gene and its expression in MSCs provide a sound basis for the combination of VEGF-B gene therapy and autologous MSCs transplantation in ischemic heart disease.

关 键 词：真核表达载体 血管内皮生长因子B 骨髓间质干细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]