Ikuno Yasushi, Kazlauskas Andrius
Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
Invest Ophthalmol Vis Sci. 2002 Jul;43(7):2406-11.
Proliferative vitreoretinopathy (PVR) is a serious problem in vitreoretinal surgeries. A report of a previous study has indicated that platelet-derived growth factor alpha receptor (alphaPDGFR) plays an important role in a rabbit model of this disease and that a dominant negative alphaPDGFR potently suppresses PVR in an ex vivo setting. Herein, the effect of in vivo gene delivery of a dominant negative alphaPDGFR on PVR was tested in a rabbit model of the disease.
The dominant negative alphaPDGFR (TalphaR) is a truncated version of the receptor, which does not have the intracellular domain. It was expressed by using a retrovirus. In vitro characterization of TalphaR was performed in primary cultures of rabbit conjunctival fibroblasts (RCFs). Western blot analysis was used to check the expression of TalphaR protein. A type I collagen gel contraction assay was performed to test the efficacy of TalphaR on PDGF-dependent cellular responses in vitro. The in vivo efficacy and specificity of the retrovirus was determined by injecting a green fluorescent protein (GFP) retrovirus into rabbits that had been preinjected with RCFs. The impact of the TalphaR retrovirus on PVR was tested by using the rabbit model in which PVR was induced by the injection of RCFs and platelet-rich plasma (PRP).
TalphaR was expressed at more that 50 times the level of endogenous alphaPDGFR in RCFs and severely reduced PDGF-dependent contraction of collagen gels. Intravitreal injection of the GFP retrovirus resulted in expression of GFP primarily in the injected RCFs. Whereas injection of RCFs induced complete retinal detachment in 100% of the animals, co-injection of the TalphaR retrovirus substantially reduced the severity and incidence of retinal detachments.
Gene therapy with a retrovirus used to express a dominant negative alphaPDGFR attenuates PVR in a rabbit model of the disease. This strategy may be a new approach to preventing PVR in humans.
增殖性玻璃体视网膜病变（PVR）是玻璃体视网膜手术中的一个严重问题。先前一项研究报告表明，血小板衍生生长因子α受体（αPDGFR）在该疾病的兔模型中起重要作用，并且一种显性负性αPDGFR在体外环境中能有效抑制PVR。在此，在该疾病的兔模型中测试了显性负性αPDGFR的体内基因递送对PVR的影响。
显性负性αPDGFR（TαR）是该受体的截短版本，不具有细胞内结构域。它通过逆转录病毒表达。在兔结膜成纤维细胞（RCF）的原代培养物中对TαR进行体外特性分析。采用蛋白质印迹分析来检测TαR蛋白的表达。进行I型胶原凝胶收缩试验以测试TαR在体外对血小板源性生长因子（PDGF）依赖性细胞反应的功效。通过将绿色荧光蛋白（GFP）逆转录病毒注射到预先注射了RCF的兔体内来确定逆转录病毒的体内功效和特异性。通过使用注射RCF和富血小板血浆（PRP）诱导PVR的兔模型来测试TαR逆转录病毒对PVR的影响。
TαR在RCF中的表达水平是内源性αPDGFR的50倍以上，并严重降低了胶原凝胶的PDGF依赖性收缩。玻璃体内注射GFP逆转录病毒导致GFP主要在注射的RCF中表达。虽然注射RCF在100%的动物中诱导了完全性视网膜脱离，但同时注射TαR逆转录病毒可显著降低视网膜脱离的严重程度和发生率。
用逆转录病毒进行基因治疗以表达显性负性αPDGFR可减轻该疾病兔模型中的PVR。这种策略可能是预防人类PVR的一种新方法。
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