人类细胞外囊泡的产量优化与应用  

Optimization of Production and Application of Human Extracellular Vesicles

作　　者：王晨蕾 程临钊 刘森泉 WANG Chenlei;CHENG Linzhao;LIU Senquan(Division of Life Sciences and Medicine,University of Science and Technology of China,Hefei 230027,China)

机构地区：[1]中国科学技术大学生命科学与医学部,合肥230027

出　　处：《中国生物工程杂志》2024年第9期1-13,共13页China Biotechnology

基　　金：国家自然科学基金(82250710171、82100243)资助项目。

摘　　要：目的:构建与优化细胞外囊泡(extracellular vesicles,EVs)的高产平台,验证此平台是否适用于功能蛋白的装载与递送。方法:通过实时荧光定量PCR(qRT-PCR)、流式细胞术或Western blot(WB)检测细胞目的基因表达,采用差速超速离心法浓缩EVs,分别利用WB与纳米颗粒跟踪分析(nanoparticle tracking analysis,NTA)检测EVs标记蛋白表达、粒径分布与产量。利用脂多糖(lipopolysaccharides,LPS)诱导的A549细胞损伤模型验证EVs递送功能蛋白的能力。通过CCK8检测细胞活力,通过Annexin V检测细胞凋亡,利用qRT-PCR检测Wnt/β-catenin下游基因AXIN2、TCF4、NKD1的表达。结果:流式细胞术结果表明CD9、CD81均能在HEK293T和HEK293F上过表达,NTA检测显示CD9更有助于EVs产量的提高。实现在贴壁细胞体系中EVs产量提升4倍,在悬浮细胞体系中EVs产量提升6倍。之后验证高产平台是否适用于功能蛋白Wnt3a的装载与递送。WB结果证实此高产平台能装载Wnt3a蛋白,NTA检测表明此高产平台在装载Wnt3a的同时将EVs产量提升3倍左右。CCK8、Annexin V结果显示CD9 Wnt3a EVs能明显提高A549细胞在LPS诱导损伤后的细胞活力,抑制细胞凋亡。机制上,qRT-PCR表明CD9 Wnt3a EVs激活了Wnt/β-catenin下游基因AXIN2、TCF4、NKD1的表达。结论:构建了一种基于CD9过表达的EVs高产平台,该平台对EVs产量的提升在贴壁细胞与悬浮细胞中均得到了验证,该平台对功能蛋白Wnt3a的递送能力在LPS诱导的A549细胞损伤模型中得到了验证。该平台为实现EVs量产提供了新策略,有望成为一种新的功能蛋白递送平台,在生物学研究和无细胞治疗领域开拓更多应用。Objective:To optimize the production and application of extracellular vesicles(EVs)for protein delivery through the use of genetic engineering.Methods:qRT-PCR,flow cytometry and Western blot(WB)were applied to determine the expression of target genes in producer cells.EVs were isolated and purified from the conditioned medium derived from engineered cells by sequential ultracentrifugation.The particle concentration and size distribution of EVs were measured by nanoparticle tracking analysis.The representative EV markers were characterized by WB.The therapeutic effect of EVs was estimated in a human alveolar epithelial cell injury model.Cell viability was determined by CCK8 assay,cell apoptosis was assessed by Annexin V,and expression of Wnt/β-catenin downstream genes such as AXIN2,TCF4,and NKD1 was tested by qRT-PCR.Results:CD9 or CD81 overexpression was confirmed by flow cytometry in HEK293T and HEK293F.CD9 was superior to CD81 in promoting EV production.Moreover,CD9 tripled the yield of EVs in both adherent cells and suspension cells.We also investigated whether this platform could be used to deliver proteins such as Wnt3a.The results showed that Wnt3a was highly loaded on EVs and that CD9 Wnt3a EVs improved the cell viability and reduced the apoptosis after cell injury.Conclusions:We have developed a novel platform that can enhance the production of EVs based on CD9 overexpression.In addition,this approach is also compatible with protein delivery,providing a potential cell-free therapeutic option for clinical applications.

关 键 词：细胞外囊泡 CD9 CD81 WNT3A 

分 类 号：Q-33[生物学]