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放射性标记针对荧光素酶报告基因(Luc)的siRNA,与不同分子量的壳聚糖(CS)制备成CS/siRNA复合物,转染可稳定表达Luc基因的MDA-MB-231/Luc人乳腺癌细胞系.与较大分子量壳聚糖相比,低分子量壳聚糖(LMWC)与siRNA形成的复合物具有更小的粒径及更强的siRNA转染能力,但是对靶基因的沉默效应却不高.其原因归咎于低分子量壳聚糖(Mr为2 000或5 000,LMWC)与siRNA间强烈的电荷引力限制了siRNA在细胞内的释放.合成基序为LLLRRRDNEY*FY*VRRLL的可磷酸化短肽(p SP)与LMWC相偶联,合成p SP-LMWC.分别对siRNA及p SP-LMWC进行FAM及Dabcyl标记,利用FRET技术检测细胞内p SP-LMWC与siRNA的解离.结果表明,p SP修饰可大幅度增加siRNA与LMWC在细胞内的解离,成为有功能的游离形式,从而显著下调靶基因的表达.本文的结果表明,低分子量壳聚糖具有良好的siRNA递送能力,促进其与siRNA在细胞内的解离可有效提高siRNA对靶基因的沉默效应.
The siRNA against luciferase reporter gene (Luc) was radioactively labeled with chitosan (CS) of different molecular weights to prepare a CS / siRNA complex and the MDA-MB-231 / Luc human breast cancer Cell lines.Compared with the larger molecular weight chitosan, LMWC and siRNA have smaller size and stronger siRNA transfection ability, but the silencing effect on the target gene The reason for this is due to the fact that the strong charge attraction between low molecular weight chitosan (Mr 2 000 or 5 000, LMWC) and siRNA limits the intracellular release of siRNA.The synthetic motif is LLLRRRDNEY * FY * VRRLL The phosphorylated short peptide (p SP) was coupled with LMWC to synthesize p SP-LMWC.The FAM and Dabcyl labeling of siRNA and p SP-LMWC were respectively carried out, and the dissociation of p SP-LMWC and siRNA was detected by FRET technique. The results show that pSP modification can significantly increase the intracellular dissociation of siRNA and LMWC, and become a functional free form, thus significantly downregulating the target gene expression.The results of this paper show that low molecular weight chitosan has good siRNA delivery Ability to promote its dissociation with siRNA in the cell can effectively improve the siRNA target gene sink Mo effect.