番茄外泌体的分离提取工艺优化及其作为药物载体的可行性分析  

Optimization of tomato-derived exosomes isolation and analyzsis of feasibility of it as nano carriers

作　　者：吴菊萍 肖倩 王建国[1] 王莺[1] Ju-ping Wu;Qian Xiao;Jian-guo Wang;Ying Wang(Cancer Research Institute,School of Basic Medical Science,Southern Medical University,Guangzhou,Guangdong 510515,China)

机构地区：[1]南方医科大学基础医学院肿瘤研究所

出　　处：《中国现代医学杂志》2019年第24期8-14,共7页China Journal of Modern Medicine

基　　金：2014年广州市科技攻关项目(No:2014J4100149);2015年南方医科大学创新驱动项目(No:CX2015N004)

摘　　要：目的优化番茄外泌体提取工艺,初步探索番茄外泌体作为纳米载体的可行性。方法利用蔗糖密度梯度法提取番茄外泌体;动态光散射仪和透射电镜鉴定外泌体纳米粒子特征;用MTT法检测外泌体对A549、A2780及HONE1(EBV+)3种肿瘤细胞的毒性。构建外泌体携带模型药物DiI和目的基因反义EBV-miR-BART7-3p的纳米药物,考察其细胞摄取能力,实时荧光定量聚合酶链反应检测基因药物的沉默效果。结果优化工艺可获得具有纳米粒径的高收率外泌体(1.31 g/kg),呈球形脂质双分子层结构,并对细胞无毒性。外泌体构建的纳米药物可被细胞摄取并分布于胞浆。外泌体负载基因可有效降低目的基因的表达水平(P<0.05)。结论多步差速离心法和蔗糖密度梯度法可优化番茄外泌体的提取工艺,番茄外泌体可作为优良药物载体。Objective To optimize extraction process of tomato-derived exosomes and preliminarily exploration of the feasibility of tomato-derived exosomes as nano carriers.Methods Tomato-derived exosomes were isolated by sucrose density gradient method.Dynamic light scattering and transmission electron microscopy were used to identify the characteristics of exosomes,and MTT assay was used to detect the toxicity of exosomes in A549,A2780 and HONE1(EBV+)cells.Tomato-derived exosomes were used to deliver model drug DiI and antisense EBV-miR-BART7-3p to prepare nanomedicines.Their cellular uptake ability was tested in three cancer cells and the silence effect of nano gene drugs was investigated by the fluorescent quantitative PCR.Results Exosomes with high yield(1.31 g/kg)were obtained by the optimized procedure.Tomato-derived exosomes exhibited nano-size and showed the spherical lipid bilayer structure,and they had no significant cytotoxicity.The nanomedicine based on exosomes could be internalized in cells and distributed in the cytoplasm.The targeted gene expression level could be effectively silenced by gene drug,and the difference was statistically significant(P<0.05).Conclusion The extraction procedure of tomato-derived exosomes was optimized,and the feasibility of exosomes as excellent drug carriers was verified.

关 键 词：番茄 外泌体 药物载体 

分 类 号：R733[医药卫生—肿瘤]