Ren Jian-Li, Wang Zhi-Gang, Zhang Yong, Zheng Yuan-Yi, Li Xing-Sheng, Zhang Qun-Xia, Wang Zhao-Xia, Xu Chuan-Shan
Institute of Ultrasound Imaging, The Second Affiliated Hospital of Chongqing Medical University, Chongqing City, China.
Ultrasound Med Biol. 2008 Nov;34(11):1857-67. doi: 10.1016/j.ultrasmedbio.2008.03.019. Epub 2008 Jun 4.
The aim of the present study was to explore the gene transfection efficiency of Tat peptide/plasmid DNA/ liposome (TDL) compound combined with ultrasound-targeted microbubble destruction (UTMD) in human umbilical vein endothelial cell (HUVEC). Tat peptide, plasmid DNA (pIRES2-EGFP-HGF) and Lipofectamine 2000 were used to prepare the TDL compound. Microbubbles were prepared using mechanic vibration. The expression of the report gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The viability of HUVEC was measured by MTT assay. mRNA and protein of HGF was analyzed by reverse transcription-polymerase chain reaction and Western Blot. The intensity of green fluorescence and the gene transfection efficiency of TDL compound + microbubbles + ultrasound group were higher than those of other groups, and no significantly different viability was found between TDL compound + microbubbles + ultrasound group and the other groups. The HGF mRNA and HGF protein of TDL compound + microbubbles + ultrasound group were higher than those of other groups. Our finding demonstrated that UTMD could enhance the transfection efficiency of TDL compound without obvious effects on the cell viability of HUVEC, suggesting that the combination of UTMD and TDL compound might be a useful tool for the gene therapy of ischemic heart disease.
本研究的目的是探讨Tat肽/质粒DNA/脂质体（TDL）复合物联合超声靶向微泡破坏（UTMD）对人脐静脉内皮细胞（HUVEC）的基因转染效率。使用Tat肽、质粒DNA（pIRES2-EGFP-HGF）和Lipofectamine 2000制备TDL复合物。采用机械振动法制备微泡。使用荧光显微镜和流式细胞术观察报告基因增强绿色荧光蛋白（EGFP）的表达。采用MTT法检测HUVEC的活力。通过逆转录-聚合酶链反应和蛋白质免疫印迹法分析HGF的mRNA和蛋白。TDL复合物+微泡+超声组的绿色荧光强度和基因转染效率高于其他组，且TDL复合物+微泡+超声组与其他组之间的细胞活力无显著差异。TDL复合物+微泡+超声组的HGF mRNA和HGF蛋白高于其他组。我们的研究结果表明，UTMD可提高TDL复合物的转染效率，且对HUVEC的细胞活力无明显影响，提示UTMD与TDL复合物联合应用可能是缺血性心脏病基因治疗的一种有效手段。
