阳离子脂质体介导小鼠原代螺旋神经节细胞NT-3基因转染的实验研究

Cationic liposome mediated NT-3 gene to transfect mice spiral ganglion cells

目的通过阳离子脂质体携带构建的神经营养因子-3(NT-3,Neurotrophin-3)-绿色荧光蛋白基因(pEGFPC2)转染小鼠耳蜗原代培养的螺旋神经节细胞,观察NT-3-pEGFPC2的共表达及其对螺旋神经节细胞生长的影响。方法构建携带绿色荧光蛋白报告基因的重组质粒-NT3 pEGFPC2,建立体外培养原代小鼠耳蜗螺旋神经节细胞的方法,并用神经丝蛋白(NF200)单克隆抗体进行免疫组化染色鉴定。利用阳离子脂质体携带重组质粒瞬时转染原代小鼠耳蜗螺旋神经节细胞,观察转染后NT-3的表达及对螺旋神经节细胞生长数量的影响。结果成功培养了小鼠原代耳蜗螺旋神经节细胞。基因转染后24 h在荧光显微镜下观察到胞体及轴突发出绿色荧光的螺旋神经节细胞。用Western blot方法检测到相应NT-3蛋白表达。继续培养12 d后各组细胞数量均减少,但NT-3转染组细胞减少数量低于空载体组。结论阳离子脂质体可作为NT-3基因的载体。NT-3的基因转染对抑制耳蜗螺旋神经节细胞的退行性变有一定作用。

Objective To observe the localized expression of NT-3-pEGFPC2 and the influence to the growth of spiral ganglion cells （SGCs） by cationic liposome with the gene-NT-3-pEGFPC2 transfecting mice arche-generation spiral ganglion cells. Methods The eukaryonic expression vector （NT-3-pEGFPC2） with the report gene-pEGFPC2 was constructed. The SGCs were cultured and purged in vitro and identified by neurofilament （NF200） monoclonal antibody immunochemical staining. The NT-3-pEGFPC2 was transiently transfected into SGCs by using lipofectamine as the vector. The expression of NT-3 after transfection and the influence to the number of SGCs were observed. Results The SGCs were successfully cultured and identified. The cell bodies and axons of SGCs with green fluorescence were observed by fluorescence microscope. The expression of NT-3 was checked by the Western blot. The decreasing number of SGCs in the group transfected with NT-3 was less than that in the control group after twelve days. Conclusion Cationic liposome can be a vector of NT-3 in gene therapy. The degradation of SGCs can be alleviated by gene transfection of NT-3 in vitro.