Department of Ophthalmology, Second Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing, China.
Chin Med J (Engl). 2009 Nov 20;122(22):2711-7.
Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization (CNV), and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor (PEDF) could inhibit CNV.
Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDF gene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure (intravitreal injection); infusing liposomes with the naked plasmid DNA of PEDF into the vitreous (lipofectamine + PEDF); infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately (retrobular injection); infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately (vein injection). The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.
In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively.
Ultrasound-microbubble technique could increase PEDF gene transfer into rats' retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.
许多研究表明，血管生成因子和抗血管生成因子表达的失衡对脉络膜新生血管（CNV）的发展有重要贡献，超声微泡联合系统可以成功提高基因转染效率。本研究旨在探讨超声介导的微泡破坏是否能有效地将治疗性质粒转染到大鼠视网膜中，以及色素上皮衍生因子（PEDF）的基因转移是否能抑制 CNV。
分离培养人视网膜色素上皮细胞，分别用超声、质粒或质粒联合超声和微泡处理，探讨微泡增强超声增强 PEDF 基因表达的可行性；对于体内动物研究，采用氩激光诱导大鼠 CNV。这些大鼠被随机分为五组，通过玻璃体注射携带 PEDF 裸质粒 DNA 的微泡，然后立即进行超声照射（玻璃体注射）；玻璃体注射携带 PEDF 裸质粒 DNA 的脂质体（脂质体+PEDF）；立即用超声照射眼眶内的微泡（视网膜后注射）；立即用超声照射股静脉内的微泡（静脉注射）。未接受任何治疗的 CNV 大鼠作为对照。治疗后 7、14 和 28 天处死大鼠并眼球摘出。通过实时定量 RT-PCR、Western blot 和免疫荧光染色分别检测 PEDF 的基因和蛋白表达。通过荧光素眼底血管造影检查 PEDF 基因转染对 CNV 的影响。
体外细胞实验表明，与微泡或超声单独作用相比，超声辐照微泡可显著增强 PEDF 的传递。在大鼠 CNV 模型中，超声/微泡介导的转染效率在治疗后 28 天显著高于脂质体介导的基因转染。研究还表明，通过超声介导的微泡破坏给药，可有效抑制大鼠 CNV。
超声微泡技术可提高 PEDF 基因向大鼠视网膜和脉络膜的转染效率，并显著抑制 CNV 的发展，提示这种非侵入性基因转移方法可能为临床基因治疗提供一种有用的工具。
