脂质体介导RNAi的研究  

Research on Cationic Vector-mediated RNAi

作　　者：张旭[1,2] 丁会芹[2,3] 王冰[2] 崔韶晖[2] 赵轶男[2] 金文实[1,2] 张树彪[2] 金梅[1] 

机构地区：[1]辽宁师范大学生命科学学院,大连116029 [2]大连民族学院生物化学工程国家民委-教育部重点实验室,大连116600 [3]中国医科大学基础医学院,沈阳110001

出　　处：《生物医学工程学杂志》2012年第4期722-726,共5页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(20876027;21046008);教育部新世纪优秀人才支持计划项目资助(NCET-08-0654);中央高校基本科研业务费专项资金资助项目(DC10020103)

摘　　要：本文选用阳离子脂质体Lipofectamine 2000为基因载体,使用沉默荧光素酶基因的小干扰RNA(siRNA)进行体外RNA干扰(RNAi),对阳离子脂质体转运siRNA的效率进行研究。首先以阳离子脂质体运载质粒DNA转染细胞,并以延滞实验对阳离子脂质体与siRNA的结合能力进行检测;然后通过基因载体将荧光素酶基因载入细胞,转运不同浓度的siRNA对其进行沉默,应用酶标仪对荧光素酶基因的表达进行分析,并用MTT法对转染后细胞存活率进行检测。结果表明:阳离子脂质体Lipofectamine 2000可以高效转运质粒DNA,并可与siRNA很好地结合。在较低的siRNA浓度下,对荧光素酶基因的沉默率达到较高的水平,转运siRNA转染细胞对细胞活性的影响很小,但细胞存活率随着siRNA浓度的增加而逐渐降低。通过优化实验条件,阳离子脂质体转运较低浓度的siRNA即可高效沉默目的基因。In order to study the efficiency of small interfering RNA（siRNA） transfer mediated by cationic liposome, we used luciferase siRNA to evaluate the gene silencing activity in the Hep-2 cells, which were stably transduced with a luciferase gene. The pDNA transfection was studied, and siRNA arrearage assay was conducted to determine the capability of cationic liposome with siRNA. Different concentrations of siRNA was used to silence luciferase gene＇ s activity, and then the result was examined by microplate reader. Cell viability was analyzed after transfection by MTT assay. The results suggested that Lipofectamine 2000 could transfer the pDNA efficiently, and have strong binding capacity with siRNA. The silencing efficiency of luciferase was obtained with low concentration of siRNA. The cell viability was influenced by RNA interference （RNAi） very slightly, but the cell survival rate decreased with the increase of siRNA concentrations. It was well concluded that by optimizing the experimental conditions, cationic liposome can transfer low concentration siRNA to silence target genCs activity efficiently.

关 键 词：基因治疗 RNA干扰 阳离子脂质体 小干扰RNA LIPOFECTAMINE 2000 

分 类 号：Q78[生物学—分子生物学]