纳米颗粒跟踪分析和纳米流式细胞仪对脂肪间充质干细胞的细胞外囊泡检测结果评价

Detection Results Evaluation of Adipose Mesenchymal Stem Cells Derived Extracellular Vesicles by Nanoparticle Tracking Analysis and Nano-flow Cytometry

关键词:  脂肪间充质干细胞  细胞外囊泡  纳米颗粒跟踪分析  纳米流式细胞仪

Key words:  adipose mesenchymal stem cells  extracellular vesicles  nanoparticle tracking analysis  nano-flow cytometry

摘要:

目的 探讨纳米颗粒跟踪分析（nanoparticle tracking analysis，NTA）和纳米流式细胞仪（nano-flow cytometry，nanoFCM）对对照品微球和脂肪间充质干细胞（adipose mesenchymal stem cells，AdMSCs）的细胞外囊泡（extracellular vesicles，EVs）的检测能力，为EVs的鉴定和质量控制提供依据。方法 首先使用对照品微球来验证NTA和nanoFCM的检测能力。随后，对于聚乙二醇6000沉淀-超速离心法制备的AdMSCs-EVs，通过Western blotting和电子透射显微镜进行一般特征鉴定后，使用NTA和nanoFCM来检测其粒径分布和PKH67染色后的荧光颗粒占比。结果 NTA和nanoFCM在颗粒浓度方面的检测能力相近，但是nanoFCM的精度更高，粒径区分度更好。用NTA检测EVs的检测结果显示NTA粒径分布较广，nanoFCM粒径分布较窄；NTA的PKH67的荧光检测阳性率显著低于nanoFCM。结论 NanoFCM拥有较高的精度，但也存在着一定应用局限性。NTA的粒径区分度相对较差，但是可检测的粒径范围更广。因此，NTA可以满足EVs的粒径分布检测，对荧光检测具有较高灵敏度的nanoFCM更适合分析EVs的表面标志物比例。

Abstract:

OBJECTIVE To explore the detection capability of nanoparticle tracking analysis(NTA) and nano-flow cytometry(nanoFCM) in detecting standard microspheres and adipose mesenchymal stem cells(AdMSCs) derived extracellular vesicles(EVs), provide experimental basis for the identification and quality control of EVs. METHODS Firstly, the detection capabilities of NTA and nanoFCM were verified by detecting standard microspheres. For the AdMSCs-EVs produced via PEG6000-based ultracentrifugation method, the general characterizations were performed with Western blotting and transmission electron microscope. The particle size distributions as well as the post-PKH67 labeling positive EV ratios were analyzed by NTA and nanoFCM respectively. RESULTS NTA and nanoFCM had similar capacity regarding to particle concentration detection, while nanoFCM had higher accuracy and better particle size differentiation ability. The EVs size distribution results showed that, samples analyzed by NTA presented a wider distribution while nanoFCM presented a narrower distribution. For detection of PKH67-positive EVs, the positive ratios detected by NTA were significantly lower than by nanoFCM. CONCLUSION NanoFCM has higher accuracy along with some application limitations. NTA is relatively less capable at particle size distinction, but has a wider particle size detection range. Therefore, for detection of EVs, NTA can satisfy the needs of size distribution characterization, while nanoFCM with high sensitivity for fluorescence detection is more suitable for analyzing the proportion of surface markers in EVs.