Department of Neurosurgery, Spine and Spinal Cord Institute, Yonsei University College of Medicine, Seoul, Republic of Korea.
Spine (Phila Pa 1976). 2009 Dec 15;34(26):E952-8. doi: 10.1097/BRS.0b013e3181c4af80.
STUDY DESIGN.: An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation effect of a reporter or therapeutic gene expression by an oxygen-dependent degradation (ODD) domain in a hypoxia-inducible gene expression system with or without the erythropoietin (EPO) enhancer. OBJECTIVE.: To increase vascular endothelial growth factor (VEGF) gene expression in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer upstream of the SV promoter and an ODD domain C-terminally fused to VEGF. SUMMARY OF BACKGROUND DATA.: ODD domain plays a major role in the degradation of hypoxia-inducible factor 1alpha and has been used in a hypoxia-specific gene expression system as a post-translational regulatory factor. METHODS.: The hypoxia-inducible luciferase or VEGF plasmid was constructed using the EPO enhancer combined with or without the ODD domain. The constructed plasmid was transfected into mouse Neuro 2a (N2a) neuroblastoma cells by Lipofectamine 2000, followed by a 24-hour incubation in hypoxia or normoxia. For in vivo analysis, the naked plasmid DNA was directly injected into the injured rat spinal cord. The gene expression was evaluated by luciferase activity assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and immunofluorescence staining. RESULTS.: The EPO enhancer/ODD domain-combined hypoxia-inducible gene expression system clearly increased the expression of the reporter luciferase gene and therapeutic VEGF gene specifically under hypoxic conditions and SCI, and quickly downregulated protein expression to a very low level after reoxygenation. CONCLUSION.: These results strongly suggest the potential applicability of this EPO enhancer/ODD domain-based hypoxia-inducible gene expression system in the development of a safer and more effective VEGF gene therapy for SCI.
本研究采用体外神经缺氧模型和大鼠脊髓损伤（SCI）模型，评估在缺氧诱导基因表达系统中，报告基因或治疗基因的表达受氧依赖性降解（ODD）结构域调节的情况，该系统包含有或没有促红细胞生成素（EPO）增强子的情况下。目的：为了增加 SCI 病变中血管内皮生长因子（VEGF）基因的表达，但避免 VEGF 在正常部位的非预期过表达，我们构建了一个由 EPO 增强子上游 SV 启动子和 VEGF 端融合的 ODD 结构域组成的缺氧诱导基因表达系统。背景资料概要：ODD 结构域在缺氧诱导因子 1α的降解中起主要作用，并已被用于缺氧特异性基因表达系统中作为一种翻译后调控因子。方法：使用 EPO 增强子结合或不结合 ODD 结构域构建缺氧诱导荧光素酶或 VEGF 质粒。构建的质粒通过 Lipofectamine 2000 转染到小鼠 Neuro 2a（N2a）神经母细胞瘤细胞中，然后在缺氧或常氧条件下孵育 24 小时。用于体内分析，将裸质粒 DNA 直接注射到损伤的大鼠脊髓中。通过荧光素酶活性测定、酶联免疫吸附测定、逆转录-聚合酶链反应和免疫荧光染色评估基因表达。结果：EPO 增强子/ODD 结构域结合的缺氧诱导基因表达系统在缺氧和 SCI 条件下明显增加了报告荧光素酶基因和治疗 VEGF 基因的表达，并且在再氧合后迅速将蛋白表达下调到非常低的水平。结论：这些结果强烈表明，基于 EPO 增强子/ODD 结构域的缺氧诱导基因表达系统在开发更安全、更有效的 SCI 治疗 VEGF 基因治疗方面具有潜在的应用价值。
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