PEI-g-MPEG/白细胞介素-10基因传递系统转染背根神经节细胞的体外性能研究

探索非病毒基因载体聚乙二醇-聚乙烯亚胺共聚物(PEI-g-MPEG)介导白细胞介素-10(Interleukin-10,IL-10)体外转染原代培养背根神经节细胞(dorsal root ganglion cells,DRGs)的效果.采用本实验室设计合成的PEI-g-MPEG,与同时携带增强型绿色荧光蛋白报告基因及IL-10基因的真核表达质粒DNA(pDC316-EGFP/IL-10)形成复合物,以脂质体(lipofectamine)复合体系Lipo/pDNA为对照,通过溴乙啶(ethidiumbromide,EB)排斥实验、凝胶阻滞电泳实验、粒径与电位的测定及扫描电镜等实验方法观察PEI-g-MPEG/pDNA的复合效果.并且检测了复合物对DRGs的毒性、转染效果及IL-10的蛋白表达情况.结果表明,PEI-g-MPEG在N/P(PEI-g-MPEG所含的氮原子和质粒DNA中磷原子的摩尔比)为5时可完全复合pDNA;随着N/P的增大,PEI-g-MPEG/pDNA复合物的粒径逐渐减小,而表面电位逐渐增大;在N/P为15时报告基因转染效果和IL-10蛋白表达情况较好,复合物的形貌呈大小均一的球形.PEI-g-MPEG/IL-10基因传递系统对于神经病理性疼痛的基因治疗具有潜在应用价值.

IN VITRO EVALUATION OF INTERLEUKIN-10 GENE DELIVERY INTO DORSAL ROOT GANGLION CELLS MEDIATED BY PEI-g-MPEG

The use of non-viral carrier,polyethyleneimine-g-methoxypoly(ethylene glycol) (PEI-g-MPEG) for interleukin-10 (IL-10) gene delivery into rat dorsal root ganglion cells (DRGs) was examined in vitro.Electrophoresis,ethidium bromide exclusion,SEM,size and zeta (ζ) potential measurements were performed to characterize the nanocomplex,PEI-g-MPEG/pDNA.In addition,the cytotoxicity,transfection efficiency and IL-10 protein expression level in comparison with lipofectamine as a delivery agent were determined.Results showed that plasmid DNA was completely complexed by PEI-g-MPEG at a nitrogen-to-phosphor (N/P) ratio of 5.The PEI-g-MPEG/pDNA complexes were well-distributed and spherical with smooth surface.As the proportion of PEI-g-MPEG increased,i.e.when N/P ratio increased from 1 to 30,the size of the PEI-g-MPEG/DNA complexes decreased from 229 nm to 130 nm,while the surface charge increased from -19 mV to 32 mV,which led to the increased cytotoxicity.Flow cytometry revealed that the highest transfection efficiency (24%) using PEI-g-MPEG as a delivery agent was obtained at N/P charge ratio of 15,which was higher than that of lipofectamine (17%) at the same N/P value.After transfection,rat IL-10 expression in DRGs primary culture assessed using ELISA was observed to localize in the cytoplasm at 48 h in concordance with the result obtained for EGFP expression.These results indicated that PEI-g-MPEG could be a potential candidate for gene delivery during the therapy of neuropathic pain.