【作者】 林勇；

【导师】 王廷华； 冯忠堂；

【作者基本信息】 昆明医学院 ， 神经生物学， 2007， 硕士

【摘要】 【目的】研究有机修饰的硅纳米粒子介导生存素基因修饰嗅鞘细胞。【方法】准备纳米粒子ORMA40，透射电镜检测纳米粒子ORMA40的形态。检测Survivin质粒及纳米粒子ORMA40与Survivin质粒的复合物。培养和纯化嗅鞘细胞，用兔抗p75抗体免疫细胞化学染色鉴定嗅鞘细胞。将纳米粒子ORMA40与Survivin质粒结合成复合物后，转染嗅鞘细胞。荧光显微镜观察增强绿色荧光蛋白(EGFP)的表达。转染3天后行Survivin免疫细胞化学染色，检测生存素(Survivin)的表达。【结果】透射电镜检查证实该纳米粒子在水溶液中分散良好。经DNA酶1作用后，生存素质粒被完全降解，而生存素质粒与纳米粒子ORMA40结合者却完全得到保护。经兔抗p75抗体免疫细胞染色证实，嗅鞘细胞可以得到纯化。转染ORMA40-mSur复合物的嗅鞘细胞能表达生存素(Survivin)和增强绿色荧光蛋白(EGFP)。转染ORMA40-pSur复合物的嗅鞘细胞能表达生存素(Survivin)。【结论】纳米粒子ORMA40介导Survivin质粒转染嗅鞘细胞的方法简单有效是一种较为理想的基因转染方法。更多还原

【Abstract】 Objective: To study Survivin gene modified olfactory ensheathing cells (OECs) with the Organically modified silica (ORMOSIL) nanoparticles.Methods: After established ORMA40 nanoparticles, transmission electron microscopy was used to determine the morphology and the size of the aqueous dispersion of nanoparticles. Examination of Survivin plasmid and the complex formation of Survivin plasmid with ORMA40 nanoparticles. The olfactory ensheathing cells (OECs) of SD mouse were isolated and purified in vitro. And the olfactory ensheathing cells (OECs) were evaluated by the immunocytochemical expression of anti-rabbit p75. The Survivin plasmid was used for transfection in olfactory ensheathing cells (OECs) with ORMA40- Survivin complex suspension. Fluorescencemicroscope observed expression of enhanced green luorescene protein (EGFP). Three days later, we make an evaluation on the expression of Survivin by immunocytochemical stain.Results: Transmission electron microscopy images showed that the particles are highly monodispersed in the aqueous dispersion of nanoparticles ORMOSI.. After treatment with DNase 1, the free plasmid was completely digested, whereas the Survivin plasmids bound to the ORMA40 nanoparticles were completely protected. It was proved that olfactory ensheathing cells (OECs) could be purified by the immunocytochemical expression of anti-rabbit p75. The transfected olfactory ensheathing cells (OECs) with ORMA40-mSur complex suspension express Survivin and enhanced green luorescene protein(EGFP). The transfected olfactory ensheathing cells (OECs) with ORMA40-pSur complex suspension express Survivin.Conclusions: The results show that the nanoparticles ORMA40 mediate Survivin plasmids transfecting olfactory ensheathing cells (OECs) is simple and efficient, and is an ideal method for gene transfection.更多还原