kpcczКомплексные проблемы сердечно-сосудистых заболеванийComplex Issues of Cardiovascular Diseases2306-12782587-9537Federal State Budgetary Institution “Research Institute for Complex Issues of Cardiovascular Diseases”10.17802/2306-1278-2024-13-3-154-166kpccz-1197Research ArticleОНЛАЙН. ОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯ. Патологическая физиологияONLINE. ORIGINAL STUDIES. Pathological physiologyОСОБЕННОСТИ ПРОДУКЦИИ ВНЕКЛЕТОЧНЫХ ВЕЗИКУЛ КЛЕТКАМИ ЛИНИИ THP-1 ПРИ СТИМУЛЯЦИИ IN VITROFEATURES OF EXTRACELLULAR VESICLE PRODUCTION BY THP-1 CELLS DURING IN VITRO STIMULATIONhttps://orcid.org/0000-0001-6516-7184АкиноАртур ДаниилAquinoArthur Daniil

младший научный сотрудник научно-исследовательской лаборатории аутоиммунных и аутовоспалительных заболеваний научно-исследовательского центра неизвестных, редких и генетически обусловленных заболеваний научного центра мирового уровня «Центр персонализированной медицины», ассистент кафедры клеточной биологии и гистологии и кафедры патологической физиологии института медицинского образования федерального государственного бюджетного учреждения «Национальный медицинский исследовательский центр им. В. А. Алмазова», Санкт-Петербург, Российская Федерация

Junior Researcher at the Research Laboratory of Autoimmune and Autoinflammatory Diseases, Research Center of Unknown, Rare and Genetically Determined Diseases, World-Class Research Center “Center for Personalized Medicine”, Assistant at the Department of Cell Biology and Histology and the Department of Pathophysiology, Institute of Medical Education, Federal State Budgetary Institution “V. A. Almazov National Medical Research Center” of the Ministry of Health of the Russian Federation, St. Petersburg, Russian Federation

akino97@bk.ruhttps://orcid.org/0000-0002-8493-5211РубинштейнАртем АркадьевичRubinsteinArtem A.

лаборант-исследователь лаборатории клеточной иммунологии федерального государственного бюджетного научного учреждения «Институт экспериментальной медицины», Санкт-Петербург, Российская Федерация

Research Assistant at the Laboratory of Cellular Immunology, Federal State Budgetary Scientific Institution “Institute of Experimental Medicine”, St. Petersburg, Russian Federation

https://orcid.org/0009-0003-8873-0314ГоловкинИван АлексеевичGolovkinIvan A.

студент федерального государственного автономного образовательного учреждения высшего образования «Национальный исследовательский университет ИТМО», Санкт-Петербург, Российская Федерация

Student at the ITMO University, St. Petersburg, Russian Federation

https://orcid.org/0000-0002-4433-1640ТириковаПолина ВалерьевнаTirikovaPolina V.

студентка лечебного факультета института медицинского образования федерального государственного бюджетного учреждения «Национальный медицинский исследовательский центр им. В. А. Алмазова», Санкт-Петербург, Российская Федерация

Student at the Medical Faculty, Institute of Medical Education of the Federal State Budgetary Institution “V. A. Almazov National Medical Research Center” of the Ministry of Health of the Russian Federation, St. Petersburg, Russian Federation

https://orcid.org/0000-0002-7495-446XТрулевАндрей СергеевичTrulioffAndrey S.

кандидат биологических наук научный сотрудник лаборатории общей иммунологии федерального государственного бюджетного научного учреждения «Институт экспериментальной медицины», Санкт-Петербург, Российская Федерация

PhD, Researcher at the Laboratory of General Immunology, Federal State Budgetary Scientific Institution “Institute of Experimental Medicine”, St. Petersburg, Russian Federation

https://orcid.org/0000-0001-7204-7850КудрявцевИгорь ВладимировичKudryavtsevIgor V.

кандидат биологических наук заведующий научно-исследовательской лаборатории аутоиммунных и аутовоспалительных заболеваний научно-исследовательского центра неизвестных, редких и генетически обусловленных заболеваний научного центра мирового уровня «Центр персонализированной медицины», доцент кафедры клеточной биологии и гистологии института медицинского образования федерального государственного бюджетного учреждения «Национальный медицинский исследовательский центр им. В. А. Алмазова», Санкт-Петербург, Российская Федерация; заведующий лабораторией клеточной иммунологии федерального государственного бюджетного научного учреждения «Институт экспериментальной медицины», Санкт-Петербург, Российская Федерация

PhD, Head of the Research Laboratory of Autoimmune and Autoinflammatory Diseases, Research Center of Unknown, Rare and Genetically Determined Diseases, World-Class Research Center “Center for Personalized Medicine”, Associate Professor at the Department of Cell Biology and Histology, Institute of Medical Education, Federal State Budgetary Institution “V. A. Almazov National Medical Research Center” of the Ministry of Health of the Russian Federation, St. Petersburg, Russian Federation; Head of the Laboratory of Cellular Immunology, Federal State Budgetary Scientific Institution “Institute of Experimental Medicine”, St. Petersburg, Russian Federation

https://orcid.org/0000-0002-7577-628XГоловкинАлексей СергеевичGolovkinAleksey S.

доктор медицинских наук руководитель группы генно-клеточной инженерии Института молекулярной биологии и генетики, ведущий научный сотрудник научно-исследовательской лаборатории аутоиммунных и аутовоспалительных заболеваний научно-исследовательского центра неизвестных, редких и генетически обусловленных заболеваний научного центра мирового уровня «Центр персонализированной медицины», профессор кафедры клеточной биологии и гистологии института медицинского образования федерального государственного бюджетного учреждения «Национальный медицинский исследовательский центр им. В. А. Алмазова», Санкт-Петербург, Российская Федерация

PhD, Head of the Group of Genetic Cell Engineering, Institute of Molecular Biology and Genetics; Leading Researcher at the Research Laboratory of Autoimmune and Autoinflammatory Diseases, Research Center of Unknown, Rare and Genetically Determined Diseases, World-Class Research Center “Center for Personalized Medicine”; Professor at the Department of Cell Biology and Histology, Institute of Medical Education, Federal State Budgetary Institution “V. A. Almazov National Medical Research Center” of the Ministry of Health of the Russian Federation, St. Petersburg, Russian Federation

Федеральное государственное бюджетное учреждение «Национальный медицинский исследовательский центр имени В. А. Алмазова» Министерства здравоохранения Российской Федерации, Санкт-Петербург, Российская ФедерацияРоссияFederal State Budgetary Institution “Almazov National Medical Research Centre” of the Ministry of Health of the Russian FederationRussian FederationФедеральное государственное бюджетное научное учреждение «Институт экспериментальной медицины»РоссияFederal State Budgetary Scientific Institution “Institute of Experimental Medicine”Russian FederationФедеральное государственное автономное образовательное учреждение высшего образования «Национальный исследовательский университет ИТМО»РоссияITMO UniversityRussian FederationФедеральное государственное бюджетное учреждение «Национальный медицинский исследовательский центр имени В. А. Алмазова» Министерства здравоохранения Российской ФедерацииРоссияFederal State Budgetary Institution “Almazov National Medical Research Centre” of the Ministry of Health of the Russian FederationRussian FederationФедеральное государственное бюджетное учреждение «Национальный медицинский исследовательский центр имени В. А. Алмазова» Министерства здравоохранения Российской Федерации; Федеральное государственное бюджетное научное учреждение «Институт экспериментальной медицины»РоссияFederal State Budgetary Institution “Almazov National Medical Research Centre” of the Ministry of Health of the Russian Federation; Federal State Budgetary Scientific Institution “Institute of Experimental Medicine”Russian Federation202430092024133154166Copyright © Акино А., Рубинштейн А.А., Головкин И.А., Тирикова П.В., Трулев А.С., Кудрявцев И.В., Головкин А.С., 20242024Акино А., Рубинштейн А.А., Головкин И.А., Тирикова П.В., Трулев А.С., Кудрявцев И.В., Головкин А.С.Aquino A., Rubinstein A.A., Golovkin I.A., Tirikova P.V., Trulioff A.S., Kudryavtsev I.V., Golovkin A.S.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.nii-kpssz.com/jour/article/view/1197Основные положения

Основные положения

 

 

Цель

Цель. Количественная и качественная характеристика внеклеточных везикул (ВВ), продуцируемых клетками линии THP-1, в зависимости от использованного стимулятора и его дозы.

Материалы и методы

Материалы и методы. В исследовании использованы клетки культуры THP-1. Для стимуляции клеток применены 4-форбол-12-миристат-13-ацетат (PMA) в дозах 5, 16 и 50 нг/мл, липополисахарид (ЛПС) в дозах 0,1, 0,3 и 1,0 нг/мл и фактор некроза опухоли (ФНО) в дозах 5, 10 и 20 нг/мл. Активация клеток оценена изменением уровня экспрессии CD54, а наличие апоптоза/некроза – посредством подсчета DAPI (4′,6-диамидино-2-фенилиндол)-позитивных клеток с помощью высокочувствительной проточной цитометрии (hs-FCM). Размерность продуцируемых везикул и их количественная характеристика приведены с использованием анализа траектории наночастиц (NTA). Качественная характеристика экспрессируемых поверхностных молекулярных маркеров (CD9, CD63, CD54) представлена с помощью высокочувствительной многоцветной проточной цитометрии (hs-FCM).           

Результаты

Результаты. Стимуляция ФНО и PMA в средних и высоких дозировках приводит к нарастанию секретируемой фракции малых (30–150 нм) ВВ, экспрессирующих тетраспаниновые маркеры (CD9, CD63). Малые дозы ФНО и PMA не приводили к достоверному приросту количества ВВ. При использовании всех дозировок ЛПС отмечено некоторое количественное снижение ВВ в диапазоне 30–150 нм (малые ВВ). Однако средние и высокие дозы ЛПС вызывали достоверную селективную секрецию CD63-положительных ВВ при полном отсутствии динамики изменения CD9-позитивных везикул. Активация клеток, оцениваемая по экспрессии CD54, носила дозозависимый характер и была наиболее выражена при стимуляции PMA.

Заключение

Заключение. Каждый вид использованной стимуляции клеток THP-1 провоцирует изменение функциональной активности клеток в отношении продуцируемых ими ВВ. При этом может меняться не только количественный, но и качественный состав продуцируемых объектов. Наиболее предпочтительными в стимуляции везикуляции оказались средние и высокие дозы ФНО и PMA. Стимуляция ЛПС в средних и высоких дозах может быть использована для получения изолированной CD63-положительной фракции ВВ.

Highlights

Highlights

 

 

Aim

Aim. To quantitatively and qualitatively characterize THP-1-derived extracellular vesicles in depending on type and dose of used stimulators.

Methods

Methods. THP-1 cells were stimulated by PMA (4-phorbol 12-myristate 13-acetate), LPS (lipopolysaccharide) and TNF (tumor necrosis factor). Cell activation and viability were assessed by the expression of CD54 and DAPI (4′,6-diamidino-2-phenylindole) staining respectively. Size and quantitative characteristics were assessed using nanoparticle tracking analysis (NTA). Qualitative assessment of surface molecular markers (CD9, CD63, CD54) was performed by high-sensitivity multicolor flow cytometry (hs-FCM).

Results

Results. TNF and PMA stimulation in medium and high doses led to an increase in quantity of small EVs (30–150 nm) that express tetraspanins CD9 and CD63. Low doses of TNF and PMA did not lead to a significant increase of EVs compared to control. There was a decrease in amount of small EVs (30–150 nm) in LPS-stimulated probes. However, medium and high doses of LPS stimulated selective CD63-positive EVs secretion with absence of CD9-positive events. Cell activation assessed by CD54 expression have been dose-dependent and was most prominent under PMA stimulation.

Conclusion

Conclusion. Every type of THP-1 cell activators promotes changes in the functional activity of cells towards production of extracellular vesicles. Moreover, we have demonstrated quantitative and qualitative difference in THP-1-derived vesicles under different stimulation conditions. Medium and high doses of PMA and TNF were the most relevant in stimulation of vesiculation. LPS-stimulation could be used to selectively obtain CD63-positive fraction of EV.

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The authors declare that there are no conflicts of interest present.