阳离子聚合物介导的质粒EGFP基因逆行转染视网膜神经节细胞

Cationic polymer mediated plasmid encoding EGFP retrograde transfection in retinal ganglion cells

目的研究上丘和外侧膝状体注射阳离子聚合体/质粒复合物转染视网膜神经节细胞(RGCs)的有效性。方法将表达增强型绿色荧光蛋白(EGFP)的重组质粒和阳离子聚合物的混合溶液分别注射于Wistar大鼠双侧上丘和外侧膝状体,注射后10d灌注固定大鼠,取视网膜和视神经行冰冻切片,荧光显微镜下观察。结果荧光显微镜下见视网膜内层和视神经轴突纤维中有绿色荧光表达。结论上丘和外侧膝状体注射阳离子复合物包装的质粒可将外源性基因相对高效地转入RGCs。

Objective Gene therapy for protection of retinal ganglion cells and regeneration of optic nerve is the hot topic of retinal regeneration research, but how to transfer the exogenous gene into retinal ganglion cells is one of critical step in the gene therapy. The purpose of this research was to investigate the efficiency of anterior colliculi and lateral geniculate body injection of plasmid DNA encoding enhanced green fluorescent protein （EGFP） into retinal ganglion cells. Methods Cationic polymer and plasmid encoding EGFP（ 10 μg/μl）were mixed at a ratio of 1：1 ,and the complex were injected into the anterior collieuli and lateral geniculate body of 5 Wistar rats aged 8 - 10 weeks（weighed at 220 to 250 g）. Ten days after the injection, the rats were perfused fixation. The retinal and optic nerve frozen section were made and observed by fluorescence microscope. Results The green fluorescence of EGFP in retinal ganglion cells and axon fiber of optic nerve were detected under the fluorescence microscopy,indicating that plasmid-mediated EGFP was expressed in retinal ganglion cells by a reverse flow pattern. Conclusion Extrinsic plasmid DNA carried by cationic polymer could be effictively transferred into retinal ganglion cells by injecting into anterior colliculi and lateral geniculate body.