Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo
Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo
复制标题
超声靶向微泡破坏增强 AAV 介导的体外人 RPE 细胞和体内大鼠视网膜的基因转染
DOI：
10.1038/gt.2009.84
复制
发表时间：
2009-09-01
期刊：
影响因子：
5.1
通讯作者：
Du, L. F.
中科院分区：
文献类型：
--
作者：
Li, H. L.;Zheng, X. Z.;Du, L. F.
本研究探讨超声(US)靶向微泡破坏(UTMD)介导的rAAV2-CMV-EGFP基因对体外培养的人视网膜色素上皮(RPE)细胞和体内大鼠视网膜的有效性和安全性。在体外实验中，培养的人RPE细胞在不同条件下暴露于含有或不含MBS的US中。此外，还评价了UTMD对rAAV2-CMV-EGFP自身和细胞的影响。在体内研究中，将rAAV2-CMV-EGFP注射到有或没有MBS的大鼠视网膜下间隙，然后暴露于US，以检测基因转移。我们用体视显微镜研究了增强的绿色荧光蛋白(EGFP)在体内的表达，并用Axiovision 3.1软件进行了定量分析。苏木精-伊红染色和冰冻切片观察组织损伤和EGFP基因表达的定位。在体外实验中，优化的UTMD条件下rAAV2-CMV-EGFP的转染率显著高于对照组(P=0.000)。此外，对细胞和rAAV2-CMV-EGFP本身几乎没有细胞毒性。在体内研究中，UTMD可以安全地用于增强和加速视网膜的转基因表达。荧光表达主要位于视网膜层。UTMD是一种很有前途的视网膜基因输送方法。
This study was conducted to investigate the efficacy and safety of ultrasound (US)-targeted microbubble (MB) destruction (UTMD)-mediated rAAV2-CMV-EGFP transfection to cultured human retinal pigment epithelium (RPE) cells in vitro and to the rat retina in vivo. In the in vitro study, cultured human RPE cells were exposed to US under different conditions with or without MBs. Furthermore, the effect of UTMD on rAAV2-CMV-EGFP itself and on cells was evaluated. In the in vivo study, gene transfer was examined by injecting rAAV2-CMV-EGFP into the subretinal space of rats with or without MBs and then exposed to US. We investigated enhanced green fluorescent protein (EGFP) expression in vivo by stereomicroscopy and performed quantitative analysis using Axiovision 3.1 software. Hematoxylin and eosin staining and frozen sections were used to observe tissue damage and location of the EGFP gene expression. In the in vitro study, the transfection efficiency of rAAV2-CMV-EGFP under optimal UTMD was significantly higher than that of the control group (P=0.000). Furthermore, there was almost no cytotoxicity to the cells and to rAAV2-CMV-EGFP itself. In the in vivo study, UTMD could be used safely to enhance and accelerate the transgene expression of the retina. Fluorescence expression was mainly located in the retinal layer. UTMD is a promising method for gene delivery to the retina.