Extraction and identification of synovial tissue-derived exosomes by different separation techniques

Autor:	Liuwei Huang, Anmin Ruan, Pu Chen, Xiaozhe Zhang, Qing-Fu Wang, Jun Zhou, Yu-Feng Ma
Jazyk:	angličtina
Rok vydání:	2020
Předmět:	0301 basic medicine lcsh:Diseases of the musculoskeletal system Blotting Western Size-exclusion chromatography Polyethylene glycol Exosomes 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Microscopy Electron Transmission Western blot lcsh:Orthopedic surgery PEG ratio Osteoarthritis medicine Humans Orthopedics and Sports Medicine Particle Size 030203 arthritis & rheumatology medicine.diagnostic_test CD63 business.industry Vesicle Synovial Membrane Extracellular vesicles Molecular biology Microvesicles lcsh:RD701-811 030104 developmental biology chemistry Chromatography Gel Nanoparticles Synovial tissues Surgery Ultracentrifuge lcsh:RC925-935 business Ultracentrifugation Research Article
Zdroj:	Journal of Orthopaedic Surgery and Research, Vol 15, Iss 1, Pp 1-8 (2020) Journal of Orthopaedic Surgery and Research
DOI:	10.1186/s13018-020-01604-x
Popis:	Objective The aim of this study is to compare the efficiency of different separation techniques for extracting synovial tissue-derived exosomes. Methods The synovial tissue discarded during knee arthroscopy or total knee arthroplasty surgery was collected from the Third Affiliated Hospital of Beijing University of Chinese Medicine. Ultracentrifugation (UC), filtration combined with size exclusion chromatography (SECF), and 8% polyethylene glycol (PEG) were used to extract synovial tissue-derived exosomes. Transmission electron microscopy (TEM), nanoparticle tracer analysis (NTA), and Western Blot (WB) were used to detect the morphology, particle size, and biomarker proteins (CD9, CD63, Flotillin-1, and calnexin) of exosomes. Results The extracts of enriched round and discoid vesicles were successfully extracted with UC, SECF, and PEG. The results of TEM have shown that all three extraction methods can extract circular or elliptical vesicles with disc- and cup-shaped structures from the synovial tissue, with the diameter is about 30–150 nm. NTA suggested the main peaks of three groups of exosomes are around 100–120 nm, and the concentration of the three groups of exosomes was greater than 1 × 1010/ml. The results of WB showed that three positive protein markers (CD9, CD63, and Flotillin-1) were highly expressed in the suspension extracted by the three methods and low in the synovial tissue. However, the negative protein (calnexin) was highly expressed in synovial tissues and PEG group, while low in UC and SECF group. Conclusion Morphology, particle size, and labeled protein marker detection confirmed that UC, SECF, and PEG can extract exosomes derived from synovial tissue; UC and SECF are more recommended for the extraction of synovial tissue-derived exosomes, which provides a methodological basis for studying the function and mechanism of synovial tissue exosomes in the future.
Databáze:	OpenAIRE
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