成年日本血吸虫细胞外囊泡的分离与鉴定
Summary
在这里, 我们提出一个协议, 以描述细胞外囊泡 (电动车) 隔离在体外培养培养基从成年日本血吸虫。
Abstract
胞外囊泡 (EVs) 是由多种细胞释放到胞外微环境中的膜状囊泡。EVs 代表了异质泡的种群, 其大小范围介于40和 1000 nm 之间。积累的证据表明, 电动车在病原宿主相互作用中起着重要的调控作用。对血吸虫电动车的深入了解, 应能洞察血吸虫与宿主相互作用的机制, 从而有助于制定防治血吸虫病的新策略。在这里, 我们的目的是进一步研究电动车在血吸虫的功能, 提出了一个协议, 以隔离和特征的电动车从成年的日本血吸虫(S. 血吸虫)。使用离心结合商用外切隔离套件, 从体外培养基中分离出电动汽车。隔离的日本血吸虫EVs (SjEVs) 通常具有 100-400 nm 的直径, 其特征是透射电镜和西方印迹。PKH67 染料标记 SjEVs 的使用表明, SjEVs 是由受体细胞内部化的。总的来说, 我们的协议提供了一种从成人血吸虫隔离电动车的替代方法;隔离 SjEVs 可适用于功能分析。
Introduction
胞外囊泡 (EVs) 是一种含有多种蛋白质、脂质和核酸的小膜状囊泡的种群。最近的研究表明, 电动车在细胞通讯中起着至关重要的作用, 并参与了许多生理过程的调节, 包括细胞发育、免疫调节、血管生成和细胞迁移2, 3,4,5。累积证据表明, 电动车、循环外来体及其 miRNA 货物代表某些疾病的潜在生物标志物 6.
一些原生动物如阴道毛滴虫、锥虫和利什曼原虫, 已被证明能够分泌电动车;此外, 还发现蠕虫将 EVs 分泌到活主机7中。寄生电动车已被证明涉及感染的维护, 致病性8和免疫调节9。最近在血吸虫的研究中,日本曼氏(曼氏) 10,11和日本血吸虫12已经表明, 成年血吸虫分泌外切样的水泡, 可能是参与特定生物过程的功能规范。
迄今为止, 已有几种方法用于分离胞外囊泡, 如离心、超滤13、聚合物基试剂的使用、大小排除色谱和免疫亲和层析隔离。这些不同的方法具有各自的优点和局限性14。一般认为, 离心是囊泡分离的金标准方法。但是, 此方法受到可能的 EV 聚合14的限制。
在血吸虫中, 已报告了两种 EV 隔离的方法: 包括离心11、12、10和使用商用 EV 隔离套件16等几个阶段 (鸡蛋16,血吸虫童虫11, 成人血吸虫10, 12, 17).鉴于微泡的尺寸范围从数以千纳米到上千奈米, 我们开发了一种替代方法, 结合使用台式离心机, 超滤, 和商用 EV 隔离套件, 以隔离电动车从成年的日本血吸虫。隔离的电动车通常拥有直径 100-400 nm, 并成功地内化的接收细胞。
Protocol
Representative Results
Discussion
最近对电动汽车的研究表明, 血吸虫电动车在宿主病原体相互作用中起着重要作用3,9,12,16。为了进一步解决它们的管理功能, 必须将 EVs 与血吸虫隔离开来。在这里, 我们描述了一种 SjEV 隔离的替代方法。此方法从 100 nm 到 400 nm, 在成年的日本血吸虫中产生 SjEVs 的宽范围大小。下面的细胞摄取检?…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究部分或全部由中国国家自然科学基金 (31472187 和 31672550) 和中国科学院农业科技创新项目资助。
Materials
| Material | |||
| Total Exosome Isolation Reagent (from cell culture media) | ThermoFisher SCIENTIFIC | 4478359 | For isolating S. japonicum extracellular vesicles |
| PKH67 | Sigma-aldrich | MINI67 | For labeling Evs |
| RPMI 1640 Medium | ThermoFisher SCIENTIFIC | 11875119 | For parasite culture |
| Penicillin-Streptomycin | ThermoFisher SCIENTIFIC | 15140122 | |
| 0.22μm syringe filter | Merck | SLGP033RB | |
| SnakeSkin Dialysis Tubing | Thermo SCIENTIFIC | 68035 | |
| PEG8000 | Sangon Biotech | A100159 | |
| RIPA | Beyotime | P0013B | |
| EMD Millipore™ Immobilon™ Western Chemiluminescent HRP Substrate (ECL) | Fisher scientific | WBKLS0100 | |
| DAPI | Cell Signaling Technology | 4083 | |
| BCA kit | Beyotime | P0010 | |
| CD63 antibodies | Sangon Biotech | D160973-0025 | |
| PVDF | Bio-Rad | 1704273 | |
| Formvar/Carbon 400 mesh | Ted Pella | 01754-F | |
| Phosphotungstic Acid | Ted Pella | 19402 | |
| anti-mouse IgG-HRP | CWBIO | CW0102 | |
| NCTC clone 1469 cells | ATCC | ATCC® CCL-9.1™ | |
| FBS | HyClone | SV30087.02 | |
| Equipments | |||
| GE chemoluminescance imaging system | GE | ImageQuant LAS4000mini | |
| Transmission electron microscopy | Hitachi | H-7600 | |
| Milli-Q water | Milli-Q | Milli-Q Elix | |
| Eppendor Centrifuge | Eppendorf | Centrifuge 5804R | |
| Zetasizer Nano | Malvern | Zetasizer Nano ZS | |
| Ultracentrifuge | Beckman | Optima L-100 XP Ultracentrifuge | |
| Incubator | ESCO | CCL-107B | |
| Microscope | Zeiss | Zeiss Axin Observer Z1 | |
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