Human Saliva-Derived Exosomes: Comparing Methods of Isolation

被引:151
作者
Zlotogorski-Hurvitz, Ayelet [1 ]
Dayan, Dan [1 ]
Chaushu, Gavriel [2 ,3 ]
Korvala, Johanna [4 ,5 ]
Salo, Tuula [4 ,5 ,7 ]
Sormunen, Raija [6 ]
Vered, Marilena [1 ,8 ]
机构
[1] Tel Aviv Univ, Sch Dent Med, Dept Oral Pathol & Oral Med, IL-69978 Tel Aviv, Israel
[2] Tel Aviv Univ, Sch Dent Med, Dept Oral & Maxillofacial Surg, IL-69978 Tel Aviv, Israel
[3] Rabin Med Ctr, Dept Oral & Maxillofacial Surg, Petah Tiqwa, Israel
[4] Univ Oulu, Dept Diagnost & Oral Med, Inst Dent, SF-90100 Oulu, Finland
[5] Oulu Univ Hosp, Med Res Ctr, Oulu, Finland
[6] Oulu Univ Hosp, Dept Pathol, Bioctr Oulu, Oulu, Finland
[7] Univ Helsinki, Inst Dent, Helsinki, Finland
[8] Chaim Sheba Med Ctr, Inst Pathol, IL-52621 Tel Hashomer, Israel
关键词
saliva; extracellular vesicles; exosomes; ultracentrifugation; ExoQuick; isolation; PROTEOMIC ANALYSIS; RNA; MICRORNAS; CANCER; CD63; PROTEINS; VESICLES; LIGANDS; PLASMA;
D O I
10.1369/0022155414564219
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
ExoQuick-TCTM (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.
引用
收藏
页码:181 / 189
页数:9
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