Ogata N, Otsuji T, Matsushima M, Kimoto T, Yamanaka R, Takahashi K, Wada M, Uyama M, Kaneda Y
Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, Japan.
Curr Eye Res. 1999 Apr;18(4):261-9. doi: 10.1076/ceyr.18.4.261.5358.
The purpose of this study was to determine whether the inactivated hemagglutinating virus of Japan (HVJ)-liposome method can induce phosphorothioate oligonucleotides effectively into an experimentally-induced choroidal neovascularization of rats. We also examined whether antisense phosphorothioate oligonucleotides against VEGF could be induced into choroidal neovascularization as a therapeutic agent by the HVJ-liposome method.
The experiments were conducted on a rat model of choroidal neovascularization. FITC-labeled phosphorothioate oligonucleotides were coencapsulated in liposomes. The liposomes were coated with the envelope of inactivated HVJ and injected into the vitreous cavity following photocoagulation of pigmented rat eyes. The eyes were removed following injection, fixed, frozen and cut into thin sections. Induction of oligonucleotides was observed under a laser confocal scanning microscope for fluorescence and the development of choroidal neovascularization was evaluated histopathologically.
Phosphorothioate oligonucleotides were effectively induced into ganglion cells and into the cells of the choroidal neovascularization induced by laser photocoagulation. Highly effective induction of oligos was observed 3 to 14 days after intravitreal injection of HVJ-liposomes after which the level decreased. Antisense oligonucleotides against VEGF were induced specifically into cells in the choroidal neovascularization, however neovascularization was still observed.
Phosphorothioate oligonucleotides can be effectively induced into ganglion cells, and specifically into cells in choroidal neovascularization. Although antisense oligonucleotides against VEGF failed to prevent choroidal neovascularization, the HVJ-liposome method provided a highly effective means of inducing antisense oligos for in vivo antisense therapy.
本研究旨在确定日本灭活血凝病毒（HVJ）-脂质体法能否有效地将硫代磷酸寡核苷酸导入实验诱导的大鼠脉络膜新生血管中。我们还研究了通过HVJ-脂质体法，针对血管内皮生长因子（VEGF）的反义硫代磷酸寡核苷酸能否作为治疗剂被导入脉络膜新生血管。
实验在脉络膜新生血管大鼠模型上进行。将异硫氰酸荧光素（FITC）标记的硫代磷酸寡核苷酸共包封于脂质体中。脂质体用灭活的HVJ包膜包被，在对有色大鼠眼进行光凝后注入玻璃体腔。注射后取出眼睛，固定、冷冻并切成薄片。在激光共聚焦扫描显微镜下观察寡核苷酸的导入情况以检测荧光，并通过组织病理学评估脉络膜新生血管的发展。
硫代磷酸寡核苷酸被有效地导入神经节细胞以及激光光凝诱导的脉络膜新生血管的细胞中。在玻璃体内注射HVJ-脂质体后3至14天观察到寡核苷酸的高效导入，之后水平下降。针对VEGF的反义寡核苷酸被特异性地导入脉络膜新生血管中的细胞，但仍观察到新生血管形成。
硫代磷酸寡核苷酸可有效地导入神经节细胞，并特异性地导入脉络膜新生血管的细胞中。尽管针对VEGF的反义寡核苷酸未能预防脉络膜新生血管形成，但HVJ-脂质体法为体内反义治疗诱导反义寡核苷酸提供了一种高效手段。
