Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in organism and possess markers of producing cells. Our study was aimed at search of exosomes in the tears of healthy humans, confirmation of their nature and examination of exosome morphological and molecular-biological characteristics. The tears (110-340 ml) were collected from 24 healthy donors (aged 46-60 years); individual probes were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in electron microscope using negative staining; and they were also used for isolation and purification of the exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). The "pellets" were subjected to electron microscopy, immunolabeling. The RNA and DNA were isolated from the samples, and their sizes were evaluated by capillary electrophoresis, the concentration and localization of nucleic acids were determined. Sequencing of DNA was performed using MiSeq ("Illumina", USA), data were analyzed using CLC GW 7.5 ("Qiagen", USA). Sequences were mapped on human genome (hg19). Electron microscopy revealed in supernatants of the tears cell debris, spherical microparticles (20-40 nm), membrane vesicles and macromolecular aggregates. The "pellets" obtained after ultracentrifugation, contained microparticles (17%), spherical and cup-shaped EVs (40-100 nm, 83%), which were positive for CD63, CD9 and CD24 receptors (specific markers of exosomes). Our study showed presence of high amount of exosomes in human tears, and relation of the exosomes with RNA (size less than 200 nt) and DNA (size was 3-9 kb). Sequencing of the DNA showed that about 92% of the reads mapped to human genome.
Ékzosomy – tip vnekletochnykh vezikul, iavliaiushchikhsia perenoschikami molekuliarnykh signalov v organizme, a takzhe nesushchikh markery kletok-produtsentov. Tsel'iu dannogo issledovaniia iavilos' vyiavlenie ékzosom v sleznoĭ zhidkosti (SZh) zdorovykh liudeĭ, podtverzhdenie ikh prirody i izuchenie morfologicheskikh i molekuliarno-biologicheskikh kharakteristik. Obraztsy SZh odnokratno sobirali u 24 zdorovykh donorov (45-60 let), tsentrifugirovali pri 20000 g (15 min) dlia osazhdeniia kletochnogo debrisa. Supernatanty issledovali metodom negativnogo kontrastirovaniia, a takzhe ispol'zovali dlia vydeleniia i ochistki ékzosom (ul'trafil'tratsiia cherez pory 100 nm, dvoĭnoe ul'tratsentrifugirovanie (90 min, 100000 g, 4°C). Poluchennye obraztsy issledovali metodami élektronnoĭ mikroskopii i immunotsitokhimii, vydeliali RNK i DNK, opredeliali ikh razmer kapilliarnym élektroforezom, opredeliali kontsentratsiiu i lokalizatsiiu DNK v ékzosomakh. Sekvenirovanie DNK provodili na sekvenatore MiSeq (Illumina), dannye analizirovali s pomoshch'iu CLC GW 7.5 (CLC Bio). Posledovatel'nosti kartirovali na genom cheloveka (hg19). Supernatant SZh soderzhal kletochnyĭ detrit, sfericheskie mikrochastitsy (20-40 nm) i vezikuly, chast' kotorykh po razmeru i morfologii sootvetstvovala ékzosomam. Preparaty, poluchennye iz SZh putem ul'tratsentrifugirovaniia i ul'trafil'tratsii, soderzhali mikrochastitsy (17%), sfericheskie i chasheobraznye vezikuly (40-100 nm, 83%), na poverkhnosti kotorykh immunotsitokhimicheski byli vyiavleny spetsificheskie dlia ékzosom markery (CD63, CD9 i CD24). Provedennoe issledovanie pokazalo, chto v SZh liudeĭ soderzhatsia ékzosomy v vysokoĭ kontsentratsii, s kotorymi sviazany RNK (razmerom menee 200 par nukleotidov) i genomnaia DNK (razmerom 3-9 t.p.o.). Sekvenirovanie DNK pokazalo, chto okolo 92% prochteniĭ (reads) kartiruiutsia na genom cheloveka.
Keywords: DNA; electron microscopy; exosomes; extracellular vesicles; tears.