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维生素E脂质体纳米颗粒转运siRNA抑制丙型肝炎病毒核心蛋白表达的实验研究
Vitamin E-coupled nanoparticles-delivered siRNA inhibits expression of hepatitis C virus core protein
in vitro - 第三军医大学学报 2017年39卷第20期 页码：1980-1985
- 作者机构：
1.400010 重庆, 重庆医科大学附属第二医院: 科研处
2.400010 重庆, 重庆医科大学附属第二医院: 检验科
3.400010 重庆, 重庆医科大学附属第二医院: 感染科
- 作者简介：
[黄英, E-mail: 418face@163.com HUANG Ying, E-mail:418face@163.com
- 基金信息：国家自然科学基金面上项目(81171628)
DOI：10.16016/j.1000-5404.201705123
中图分类号： R373.21;R394.6;R977.25收稿：2017-05-31，
修回：2017-07-18，
纸质出版：2017-10-30
- 稿件说明：
移动端阅览
李小平, 颜艳, 陈红, 等. 维生素E脂质体纳米颗粒转运siRNA抑制丙型肝炎病毒核心蛋白表达的实验研究[J]. 第三军医大学学报, 2017,39(20):1980-1985.
Xiaoping LI, Yan YAN, Hong CHEN, et al. Vitamin E-coupled nanoparticles-delivered siRNA inhibits expression of hepatitis C virus core protein
in vitro [J]. Journal of Third Military Medical University, 2017, 39(20): 1980-1985.李小平, 颜艳, 陈红, 等. 维生素E脂质体纳米颗粒转运siRNA抑制丙型肝炎病毒核心蛋白表达的实验研究[J]. 第三军医大学学报, 2017,39(20):1980-1985. DOI： 10.16016/j.1000-5404.201705123.
Xiaoping LI, Yan YAN, Hong CHEN, et al. Vitamin E-coupled nanoparticles-delivered siRNA inhibits expression of hepatitis C virus core protein
in vitro [J]. Journal of Third Military Medical University, 2017, 39(20): 1980-1985. DOI： 10.16016/j.1000-5404.201705123.
摘要
目的
2
研究携带针对丙型肝炎病毒(hepatitis C virus
HCV)RNA的小干扰RNA(siRNA)的维生素E(Vitamin E)脂质体纳米颗粒在体外靶向抑制HCV复制的作用。
方法
2
采取“薄膜水化法”制备Vitamin E脂质体纳米颗粒，应用激光粒度分析仪测量纳米颗粒VE-DC/siRNA的颗粒大小及zeta电位，应用透射电子显微镜观察VE-DC形态及分散性。以Huh7.5.1细胞株作为载体。CCK-8法检测纳米颗粒的细胞毒性，琼脂糖凝胶电泳检测纳米颗粒携带siRNA的血清稳定性，蛋白印迹技术和免疫荧光技术检测该siRNA纳米颗粒的转染效率及对HCV的抑制作用。
结果
2
Vitamin E脂质体纳米颗粒形态呈球形，粒度分布为(125.5±9.0) nm，zeta电位为(39.1±4.8) mV，分散均一。相较同浓度商品化脂质体，VE-DC/siRNA对细胞的毒性相对较小。血清稳定性实验显示，VE-DC/siRNA在24 h后siRNA无降解，较裸siRNA稳定性显著提高。携带siRNA的Vitamin E脂质体纳米颗粒可以较高效率进入Huh7.5.1细胞内，并抑制HCV核心蛋白的表达。
结论
2
Vitamin E脂质体纳米颗粒可以转运siRNA至Huh7.5.1细胞，并抑制HCV核心蛋白的表达。
Abstract
Objective
2
To study the inhibitory effect of small interfering RNAs (siRNAs) targeting hepatitis C virus (HCV) delivered by vitamin E-coupled nanoparticles on the viral replication
in vitro
.
Methods
2
Vitamin E-coupled nanoparticles were prepared by thin-film hydration method. The size and surface charge (zeta potential) of the VE-DC/siRNAs nanoparticles were determined with a laser particle size analyzer. The homogeneity and dispersivity of the nanoparticles were analyzed by transmission electron microscopy. CCK-8 assay was used to assess the cytotoxicity of VE-DC/siRNAs. The stability of the nanoparticles was determined by agarose gel electrophoresis. The transfection efficiency and the inhibition of HCV was analyzed with Western blotting and immunofluorescent staining.
Results
2
The VE-DC/siRNAs nanoparticles were in spherical shape and homogeneous distribution
with their size distribution of 125.5±9.0 nm and zeta potential of 39.1±4.8 mV. These nanoparticles were characterized for their lower cytotoxicity and higher stability. The CCK-8 assay data showed that cell viability was higher for VE-DC/siRNAs treated cells than those treated with commercial liposomes at the same concentration. In serum
siRNA was intact in the VE-DC/siRNAs nanoparticles after 24 h
which was degraded in the naked siRNA within 6 h. TheVE-DC/siRNAs nanoparticles were efficiently taken up by hepatoma cells(huh7.5.1-HCV
huh7.5.1-core) observed under fluorescence microscope. Western blot analysis detected decreased levels of HCV nonstructural protein 3 (NS3) and HCV-core protein in the VE-DC/siRNAs treated hepatoma cells.
Conclusion
2
Targeted delivery of siRNAs by vitamin E-coupled nanoparticles effectively inhibits HCV replication and core protein production
suggesting that it may potentially use as delivery vehicle for target therapy.
关键词
Keywords
references
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