Wan Caifeng, Qian Jin, Li Fenghua, Li Hongli
Department of Ultrasound, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P.R. China.
Department of Ophthalmology, The Baoshan Branch Institute of Shanghai Shuguang Hospital, Shanghai 201900, P.R. China.
Mol Med Rep. 2015 Aug;12(2):2835-41. doi: 10.3892/mmr.2015.3703. Epub 2015 Apr 29.
The aim of the present study was to investigate the efficacy and safety of ultrasound-targeted microbubble destruction (UTMD)-mediated polyethylenimine (PEI) transfection in cultured human retinal pigment epithelial (RPE) cells in vitro and rat retinas in vivo. An enhanced green fluorescent protein plasmid (pEGFP) was incubated with PEI to prepare a cationic complex (PEI/pEGFP), which was confirmed using a gel retardation assay. In the in vitro study, cultured human RPE cells were subjected to US waves under different conditions with or without microbubbles. The effect of UTMD on the viability of the cells was evaluated. In the in vivo study, gene transfer was examined by injecting PEI/pEGFP into the subretinal space of the rats. The rats treated with PEI/pEGFP and UTMD served as the experimental group, while rats treated with PEI/pEGFP alone served as the control group. The transfected tissue was visualized using an inverted fluorescence microscope. The expression of EGFP was classified into three groups, negative, weak positive and strong positive. Hematoxylin and eosin staining of frozen sections was used to observe tissue damage and the location of the EGFP gene expression. The electrophoresis experiment revealed that PEI treatment was able to condense DNA efficiently. In the in vitro study, the gene transfer efficiency under the optimal UTMD condition was enhanced and significantly higher than control groups. In the in vivo study, UTMD was able to enhance transgene expression in the retina without marked tissue damage. Frozen sections of the optic cups exhibited pEGFP‑positive cells, predominantly distributed in the retina. This noninvasive novel combination of UTMD with PEI was able to enhance targeted gene delivery and gene expression in the rat retina without causing any apparent tissue damage, and may be a safe method to transfer genes and drug treatments directly to the retina, therefore being of potential therapeutic value.
本研究的目的是在体外培养的人视网膜色素上皮（RPE）细胞和体内大鼠视网膜中，研究超声靶向微泡破坏（UTMD）介导的聚乙烯亚胺（PEI）转染的有效性和安全性。将增强型绿色荧光蛋白质粒（pEGFP）与PEI孵育以制备阳离子复合物（PEI/pEGFP），并使用凝胶阻滞试验进行确认。在体外研究中，在有或无微泡的不同条件下，对培养的人RPE细胞施加超声波。评估UTMD对细胞活力的影响。在体内研究中，通过将PEI/pEGFP注入大鼠视网膜下间隙来检查基因转移。用PEI/pEGFP和UTMD处理的大鼠作为实验组，而仅用PEI/pEGFP处理的大鼠作为对照组。使用倒置荧光显微镜观察转染的组织。EGFP的表达分为三组，阴性、弱阳性和强阳性。使用苏木精和伊红对冰冻切片进行染色，以观察组织损伤和EGFP基因表达的位置。电泳实验表明，PEI处理能够有效地浓缩DNA。在体外研究中，最佳UTMD条件下的基因转移效率提高，且显著高于对照组。在体内研究中，UTMD能够增强视网膜中的转基因表达，而无明显的组织损伤。视杯的冰冻切片显示pEGFP阳性细胞，主要分布在视网膜中。这种UTMD与PEI的非侵入性新组合能够增强大鼠视网膜中的靶向基因递送和基因表达，而不会引起任何明显的组织损伤，可能是一种将基因和药物治疗直接转移到视网膜的安全方法，因此具有潜在的治疗价值。
