靶向ESCCAL_1基因的siRNA纳米复合物的制备及体外对食管癌EC-9706细胞的抑制作用

Preparation and in vitro study of ESCCAL_1-targeted siRNA gene delivery of nanocomposite for treating esophageal cancer EC-9706

目的制备装载靶向ESCCAL_1基因的siRNA纳米复合物,并考察其体外对食管癌EC-9706细胞增殖的抑制作用及对ESCCAL_1表达的影响。方法采用溶胶-凝胶法制备MSNP,通过表面修饰阳离子聚合物聚乙烯亚胺(polyethylenimine,PEI)使其带有正电荷,能够与带负电的ESCCAL_1siRNA相结合;纳米粒度仪、透射电镜测定纳米复合物的粒径和电位;凝胶电泳测定其对siRNA的包封率;MTT法检测纳米复合物体外对EC-9706细胞增殖的抑制作用;荧光显微镜观察纳米复合物中siRNA被EC-9706细胞摄取的情况;RT-PCR法检测其对EC-9706细胞中ESCCAL_1 LncRNA表达的影响。结果纳米粒度仪、透射电镜测得所合成的MSNP表面介孔直径约3~5 nm,分散性较好,尺寸较均一;能明显抑制EC-9706细胞的增殖(P<0.05),72h抑制率为(54.93±2.6)%;其装载的siRNA能被EC-9706细胞有效摄取;能有效沉默EC-9706细胞中ESCCAL_1,沉默效率为69.5%。结论采用该方法可制备包封率较高的肿瘤靶向纳米复合物,其介导的siRNA能有效抑制食管癌EC-9706细胞的体外增殖和沉默EC-9706细胞中ESCCAL_1表达。

AimTo investigate the influence of inhibitory nanocomposite on EC9706cells and the effect of nanocomposite on ESCCAL_1LncRNA expression,siRNA loaded nanocomposite being prepared as non virus delivery system MethodsMesoporous silica nanoparticles were prepared by sol gel method under room temperature and coated by cationic polymerpolyethylenimine(PEI)on the surface to stay positive charge,which could facilitate its combination with negatively charged ESCCAL_1siRNA.The size and surface charge of nanocomposite were determined by laser particle analyzer and TEM.The inhibitory rate of nanoparticles on EC9706cells was detected by MTT methods.Entrapment efficiency was determined by agarose gel electrophoresis.The uptake siRNA was detected by fluorescence microscope.The expression of ESCCAL_1LncRNA was detected by RT PCR.ResultsThe MSNP appeared to have a high dispensability and homogeneous size by particle size analyzer and transmission electron microscopy(TEM).The formed nanoparticles had a surface mesoporous diameter of3~5nm.The proliferation of ESCCAL_1was inhibited significantlly(P<005),and the72h inhibitory rate was(5493±26)%;the siRNA loading could be effectively uptaken by EC9706cells;ESCCAL_1silencing efficiency was695%.ConclusionsThe tumor targeting nanocomposite with high encapsulation efficiency is prepared.The proliferation of esophageal cancer EC9706cells can be effectively inhibited by anocomposite mediated siRNA,and the expression of ESCCAL_1is effectively silenced in EC9706cells.The nanocomposite is an efficient gene delivery system and may have potential application in gene therapy.