脂质体介导胶质细胞生长因子真核表达载体在大鼠脊髓内的表达

摘要

目的 研究脂质体介导增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和胶质细胞生长因子2(glial growth factor 2,GGF2)的双基因真核表达载体在脊髓内转染及其表达变化.方法 构建GGF2与EGFP双基因真核表达载体pIRES2-EGFP-GGF2.采用注射法将阳离子脂质体和pIRES2-EGFP-GGF2质粒混合后转染至大鼠胸段脊髓组织中.采用RT-PCR法检测不同时间点转染区域脊髓组织中GGF2 mRNA的表达.在荧光显微镜下观察不同时间点EGFP在大鼠脊髓中的表达.结果 基因转染后2d即可观察到EGFP的表达及GGF2 mRNA表达的增加,在基因转染后1～2周内EGFP及GGF2 mRNA呈高水平表达,基因转染4周后,EGFP及GGF2 mRNA的表达明显减少.结论 脂质体介导pIRES2-EGFP-GGF2转染大鼠脊髓后其表达的特点有助于基因治疗效果的即刻性,即早期大量表达,起到基因治疗的作用,又可避免外源基因的持续表达.%Objective To study the transfection and expression changes of enhanced green fluorescent protein (EGFP) and glial growth factor 2 (GGF2) double gene eukaryotic expression vector mediated by liposomes in spinal cord of rats. Methods The eukaryotic expression vector, Pires2-EGFP-GGF2, was constructed in vivo. Then a mixture of recombinant plasmid Pires2-EGFP-GGF2 and liposome was injected into the thoracic spinal cord of rats. The expression of GGF2 Mrna in the rat spinal cord was measured by reverse transcription polymerase chain reaction (RT-PCR) at different time points after gene transfection, and the expression of EGFP was observed by fluorescence microscope. Results The expressions of GGF2 Mrna and EGFP were observed in rat spinal cord 2 d after gene transfection, which were greatly increased at 1 -2 weeks after transfection and obviously decreased at 4 weeks after transfection. Conclusion The early high level expression of GGF2 Mrna and EGFP in spinal cord of rats after liposome-mediated Pires2-EGFP-GGF2 gene delivery is helpful for gene therapy, and the expression lasts only for a short time, avoiding the potential side effects of persistant expression of exogenous gene.