作　　者： ; ; ; ; ;

机构地区： 天津医科大学

出　　处： 《中国生物化学与分子生物学报》 2011年第10期980-986,共7页

摘　　要： 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. Small interfering RNA（siRNA） has been widely studied as a potential therapeutic approach for diseases with genetic defects.However,its application was largely hampered by its rapid degradation and poor cellular uptake.Chitosan（CS） nanoparticles was recently considered as a promising siRNA transporter with the advantages of low toxicity,high biodegradability and biocompatibility.Interestingly,siRNA loaded by chitosan of different molecular weight showed significantly varied silencing effect caused by target gene and its mechanism was still not well clarified.In this article,a radio-labeled siRNA,targeting firefly luciferase gene,was conjugated to chitosan of different molecular weight（varying from 2 to 80 kD） and subjected to the transfection against MDA-MB-231/Luc human breast cancer cells which stably express firefly luciferase gene.Via CS/siRNA transfection,cell permeability of the complex was evaluated by intracellular radio activity,and target gene-caused silencing effect was reflected by decreased luciferase activity.The results revealed that low molecular weight chitosan（LMWC） condensed siRNA had the highest cell permeability,but very poor silencing effect on the contrary.It implicated that the strong electrostatic interaction between LMWC and siRNA formed more condensed CS/siRNA complex to facilitate cell entrance,however,limited intracellular siRNA was unpacked to be the functional form,and as the consequence,unfavorably hindered target gene knockdown.Compared with normal LMWC,by the utilization of phosphorylatable short peptide（pSP） conjugation to promote intracellular siRNA unpacking from the LMWC carrier,target gene knockdown effect was significantly increased.In a conclusion,LMWC has the great potency to be an ideal siRNA vehicle if the issue of siRNA unpacking could be properly resolved.

关 键 词： 短肽 磷酸化 壳聚糖 解离 小干扰

领　　域： [生物学]