Department of Ophthalmology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, P R China.
Ultrasound Med Biol. 2012 Jun;38(6):1058-66. doi: 10.1016/j.ultrasmedbio.2012.02.007. Epub 2012 Apr 21.
The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells (HXO-Rb44) using ultrasound-targeted microbubble destruction (UTMD). pEGFP-C1/RB94 was transfected into HXO-Rb44 in vitro by UTMD, with liposome as the positive control. After 24 to 72 h, the expression of the reporter gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The cell viability of HXO-Rb44 was measured by a MTT assay. The mRNA and proteins of RB94, caspase-3 and Bax were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Moreover, the apoptosis rate and cell cycle progression of the cells were detected by flow cytometry. This study demonstrated that UTMD can enhance the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, suggesting that the combination of UTMD and RB94 compounds might be a useful tool for use in the gene therapy of retinoblastoma.
本研究旨在探讨超声靶向微泡破坏（UTMD）转染重组表达质粒 pEGFP-C1/RB94 到人视网膜母细胞瘤细胞（HXO-Rb44）中的作用。采用 UTMD 法，脂质体为阳性对照，将 pEGFP-C1/RB94 转染至 HXO-Rb44 细胞中，于 24 至 72 小时后，荧光显微镜和流式细胞术观察报告基因增强型绿色荧光蛋白（EGFP）的表达情况。MTT 法检测 HXO-Rb44 细胞的存活率。采用逆转录聚合酶链反应（RT-PCR）和 Western blot 分析 RB94、半胱氨酸天冬氨酸蛋白酶-3（caspase-3）和 Bax 的 mRNA 和蛋白表达。此外，采用流式细胞术检测细胞的凋亡率和细胞周期进程。本研究表明，UTMD 可增强 RB94 的转染效率，对抑制视网膜母细胞瘤细胞的生长过程有明显影响，提示 UTMD 与 RB94 化合物的联合应用可能成为视网膜母细胞瘤基因治疗的一种有效手段。
