一种siRNA药物载体薄膜的稳定性与基因沉默效应评价  

Stability and gene silencing effect of a siRNA delivery symstem

作　　者：吴丽娟[1,2] 赵芳 刘光万 曲洪媛 杨旭霞 廖囡囡[1,2] 陈丽[1,2] 杨光[1] 吴昌琳[1,2] 

机构地区：[1]华东师范大学物理系上海磁共振重点实验室,上海200062 [2]苏州博创康源生物技术有限公司,江苏苏州215300

出　　处：《中国生物制品学杂志》2017年第4期372-376,380,共6页Chinese Journal of Biologicals

摘　　要：目的分析一种siRNA药物载体薄膜在溶液中的稳定性,并评价其基因沉默效应。方法利用层层自组装(layer-by-layer self-assembly,LBL)技术制备壳聚糖(chitosan,CHS)-透明质酸(hyaluronic acid,HA)-siRNA多层薄膜。通过UV-vis吸光度法对siRNA在薄膜上的生长过程以及在不同缓释液中的稳定性分别进行验证。结合荧光图像和流式细胞术,采用直接与细胞接触的方式检测该siRNA载体薄膜的HEK293T细胞转染效应。结果 CHS-HA-siRNA多层薄膜在260 nm处有很强的吸收峰,且随着含siRNA的组装层数的增加,在260 nm的吸光度值越大。该siRNA载体薄膜在1 mol/L NaCl溶液中释放效果最好,在pH 7.4的PBS缓冲液中次之,在纯水中稳定性最好。过低或过高的盐离子浓度对促进薄膜的崩解无优势。阳性对照组7-eGFP-siRNA和2-eGFP-siRNA具有较好的沉默绿色荧光蛋白基因的效应;在细胞培养2 d后的转染试验中,阳性对照组7-eGFP-siRNA较2-eGFP-siRNA组显示出更强的基因沉默效应。结论成功构建了一种非病毒siRNA递送载体系统,为未来实现siRNA局部给药开辟了一条可行的途径。Objective To analyze the stability of a siRNA delivery system in solution and evaluate its gene silencing effect. Methods Chitosan （CHS）-Hyaluronic acid （HA）-siRNA muhilayer films were prepared through layer-by-layer self-assembly （LBL）. Both the construction and the disassembly process of siRNA-loaded films were verified by UV-vis spectroscopy. Additionally, the biological gene silencing effect of the siRNA delivery system in eGFP-HEK 293T cells was evaluated via flow cytometry and Olympus fluorescence images. Results The maximum absorption peak of siRNA-loaded muhilayer films appeared at 260 nm, and the absorbance value increased with the increasing siRNA-layer number. UV-vis spectrum verified that the siRNA-loaded films showed better disassembly performance in 1 tool/L sodium chloride solution than in PBS buffer of pH 7.4, and the highest stability was observed in pure water. Too high or too low salt ion concentration showed no advantage in the disassembly of the films. Both 7-eGFP-siRNA and 2-eGFP-siRNA multilayer films in positive control group showed good silencing effects on enhance green fluorescent protein （eGFP）. However, the transfection test 2 d after cell culture showed that the gene silencing effect of 7-eGFP-siRNA in positive control group was stronger than that of 2-eGFP-siRNA. Conclusion A non-viral siRNA delivery system was successfully constructed, which provided a feasible route for local siRNA delivery in the near future.

关 键 词：siRNA递送 层层自组装 壳聚糖-透明质酸-siRNA多层薄膜 基因沉默效应 

分 类 号：Q789[生物学—分子生物学]