壳聚糖氧化铁纳米颗粒包裹MDR1 shRNA与对胶质瘤细胞转染的影响

Chitosan-ferroferric oxide nanoparticles-encapsulated MDR1 shRNA and its effect on glioma cell transfection

目的探讨多药耐药基因短发卡RNA(MDR1 shRNA)与壳聚糖氧化铁(Fe3O4)纳米颗粒对胶质瘤细胞系转染的影响。方法设计并合成针对MDR1的shRNA序列与pSIREN-RetroQ质粒载体连接,提取质粒,制备壳聚糖纳米颗粒并包裹制备的质粒,测定包封率及粒径分布。培养人类胶质母细胞瘤BT325共转染,测定转染效率。结果质粒pSIREN-RetroQ-RNAi经双酶切后测序验证,与实验设计的shRNA长度相符。壳聚糖-铁纳米粒子的粒径为100-300 nm,分布较均匀,包裹质粒DNA包封率为97%-99%。壳聚糖Fe3O4纳米粒子包裹质粒经PI试剂转染BT325细胞,在共转染24 h开始表达,荧光显微镜下呈绿色荧光,48 h表达最强。转染效率为70%-80%(P<0.05)。结论 MDR1 shRNA表达质粒与壳聚糖Fe3O4纳米粒子结合,可提高胶质瘤细胞系BT325转染效率,转染后细胞可以正常生长,可以为进一步的靶向基因检测及治疗提供有意义的思路。

Objective To investigate the effect of encapsulated chitosan-ferroferric oxide （chitosan-Fe304） nanoparticles as carriers on the delivery efficiency ofMDR1 gene short hairpin RNA （shRNA） in human glioblastoma cell line BT325. Methods MDR1 shRNA sequence was designed and synthesized. Plasmid pSIREN-RetroQ-RNAi mixed with the MDR1 shR_NA sequence was constructed using pSIREN-RetroQ vector. Following extraction, the plasmid was encapsulated by chitosan nanoparticles, and the encapsulation rate and particle diameter distribution were measured. BT325 cell line was co-transfected with the nanoparticle complex and the optimized transfbction efficiency was determined by the ratio of GFP marker per high power field to the total number of cells in the same field. Results The shRNA plasmid vectors were constructed successfully. The diameters of the chitosan-Fe304 nanoparticles were in the range of 100-300 nm. The encapsulation rate of plasmid DNA was 97-99%. The BT325 cell line was transfected with nanoparticle complex and grew well. Gene expression was monitored using a fluorescence microscope. Expression was initiated 24 hours after transfection; peak expression was observed with a GFP marker at 48 hours. The transfection efficiency was 70-80% （P 〈 0.05）. Conclusion The combination ofMDR1 shRNA expression plasmid and chitosan-Fe304 nanoparticles can improve transfection efficiency in a glioblastoma cell line without changing the growth of the cells. This method provides innovative ideas for further target gene screening and therapy.