壳聚糖-小干扰RNA下调C6细胞多药耐药相关蛋白1表达的研究

The chitosan-small interfering RNA suppressed the expression levels of multidrug resistant-associated protein 1 in C6 cells

目的 探讨具有最大转染效率和沉默效应时壳聚糖-小干扰RNA （siRNA）的壳聚糖与siRNA（NP）比值.方法 用激光粒度仪测定壳聚糖-siRNA纳米颗粒的直径和Zeta电位.荧光标记的siRNA转染到C6细胞并初步鉴定转染效率.分6组进行转染：分别加入不同NP比值的壳聚糖-siRNA（实验组）、LipofectamineTM 2000和多药耐药相关蛋白1（MRPl） siRNA（阳性对照组）、壳聚糖-阴性对照siRNA纳米粒（阴性对照组）和空白壳聚糖纳米粒（空白组）,采用逆转录-聚合酶链反应（RT-PCR）和Western blot法检测MRPl mRNA和蛋白水平的表达.结果 成功制备了壳聚糖-siRNA纳米粒的直径和电位分别是186.7 nm和18.57 mV、209.5 nm和13.52 mV、492.3 nm和48.58 mV.其中NP比值为175时纳米颗粒对目的蛋白的表达量可以下调到（23.2±4.3）％.结论 壳聚糖-siRNA的NP比值为175时可以有效抑制MRP1基因和蛋白的表达.

Objective To investigate the chitosan-small interfering RNA （siRNA） NP ratio with maximum transfection efficiency and silence effect.Methods The diameter and Zeta potential of the chitosan-siRNA nanoparticles were measured by using laser particle size analyzer.Fluorescent-labeled siRNA was transfected into C6 cells for preliminary appraisal transfection efficiency.The transfected C6 cells were divided into 6 groups：three experimental groups,which were transfected with chitosan-siRNA nanoparticle ratio of 175∶ 1,88∶1 and 44∶ 1,the positive control group,which was transfected with multidrug resistantassociated protein 1 （MRP1）-siRNA by LipofectamineTM 2000 and MRP1 siRNA; the negative control group,which was transfected with chitosan-negative control siRNA nanoparticles,and the blank group,which was transfected with blank chitosan-nanoparticles.The expression levels of MRP1 mRNA and protein in C6 cells were assessed by using reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting respectively.Results The chitosan-siRNA nanoparticles were successfully prepared.With decreases in NP ratio,the chitosan-siRNA nanoparticles had the diameters of 186.7 nm,209.5 nm,and 492.3 am,and the potentials were 18.57 mV,13.52 mV,and 48.58 mV,respectively.Target protein expression could be down-regulated to （23.2 ± 4.3）％ when the NP ratio of nanoparticles was 175.Conclusion In C6 cells,MRP1 can be effectively suppressed when the chitosan-siRNA ratio was 175∶ 1.