装载CD47 siRNA的叶酸修饰外泌体的构建
Construction of folate-modified and CD47 siRNA-loaded exosomes
许娅楠 1厉诗娴 1吕慧中 1朱链 2许娜1
作者信息
- 1. 武汉科技大学生命科学与健康学院,湖北武汉 430065
- 2. 武汉科技大学生命科学与健康学院,湖北武汉 430065;武汉轻工大学化学与环境工程学院,湖北武汉 430023
- 折叠
摘要
[目的]构建一种装载CD47 siRNA且修饰叶酸的外泌体.[方法]将NIH/3T3细胞在含有不同浓度叶酸的培养基中培养,利用免疫荧光法检测细胞膜表面叶酸的含量,利用CCK-8法检测细胞活力.通过逐级离心分离纯化得到外泌体,通过电转将CD47 siRNA导入外泌体中,利用流式细胞术检测装载效率,通过RT-qPCR评估CD47的敲降效果.[结果]当叶酸浓度低于20 μg/mL时,NIH3T3的细胞活力未受到显著影响,且细胞膜表面富含叶酸.外泌体粒径约125 nm,膜表面富含CD9、CD63蛋白.CD47 siRNA电转外泌体的效率可达到86.9％,该外泌体将CD47 mRNA表达水平降至18％.[结论]成功构建了一种装载CD47siRNA的叶酸修饰外泌体,该外泌体可将肿瘤细胞的CD47 mRNA的表达水平下调至18％.
Abstract
[Objective]To construct folate-modified and CD47 siRNA-loaded exosomes.[Method]NIH/3T3 cells were cultured in the medium with different concentrations of folate,and the folate content was detected with immunostaining assay.The cell viability was detected by CCK-8 assay.The folate-modified exosomes were isolated by step-by-step centrifuga-tion.CD47 siRNA was loaded into the exosomes by electrotransfection.The loading efficiency was measured by flow cytometry.The knockdown effect of CD47 was evaluated by RT-qPCR.[Result]When the concentration of folate was lower than 20 µg/mL,the cell viability of NIH/3T3 was not influenced significantly and the cell membrane was rich in folate.The diameter of ex-osomes was about 125 nm,and rich in CD63 and CD9 protein expression.The electrotransfection efficiency of CD47 siRNA into exosomes was about 86.9％,and this drug-loading exosome knocked down the mRNA expression of CD47 to 18％.[Conclu-sion]CD47 siRNA loaded and folate-modified exosomes can knockdown the protein expression of CD47 of cancer cells.
关键词
叶酸/外泌体/CD47/抗肿瘤/siRNA/药物递送/纳米载体/电转Key words
folate/exosome/CD47/antitumor/siRNA/drug delivery/nanocarrier/electrotransfection引用本文复制引用
出版年
2024
NETL
NSTL
万方数据