mimmunМедицинская иммунологияMedical Immunology (Russia)1563-06252313-741XSPb RAACI10.15789/1563-0625-2019-3-427-440mimmun-1816Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESВЛИЯНИЕ NK-КЛЕТОК НА АНГИОГЕНЕЗ В УСЛОВИЯХ КОНТАКТНОГО И ДИСТАНТНОГО СОКУЛЬТИВИРОВАНИЯ С ЭНДОТЕЛИАЛЬНЫМИ КЛЕТКАМИ И КЛЕТКАМИ ТРОФОБЛАСТАNATURAL KILLER CELL EFFECTS UPON ANGIOGENESIS UNDER CONDITIONS OF CONTACT-DEPENDENT AND DISTANT CO-CULTURING WITH ENDOTHELIAL AND TROPHOBLAST CELLSМарковаК. Л.MarkovaK. L.

младший научный сотрудник лаборатории межклеточных взаимодействий, отдел иммунологии и межклеточных взаимодействий

199034, Санкт-Петербург, Менделеевская линия, 3.Тел.: 8 (812) 323-75-45, 328-98-50. Факс: 8 (812) 323-75-45.

Junior Research Associate, Cell Interactions Laboratory, Department of Immunology and Cell Interactions

199034, St. Petersburg, Mendeleevskaya line, 3. Phone: 7 (812) 323-75-45, 328-98-50. Fax: 7 (812) 323-75-45.

falcojugger@yandex.ruСтепановаО. И.StepanovaO. I.

к.б.н., старший научный сотрудник лаборатории межклеточных взаимодействий, отдел иммунологии и межклеточных взаимодействий

Санкт-Петербург

PhD (Biology), Senior Research Associate, Cell Interactions Laboratory, Department of Immunology and Cell Interactions

St. Petersburg

ШевелеваА. Р.ShevelevaA. R.

лаборант-исследователь лаборатории межклеточных взаимодействий, отдел иммунологии и межклеточных взаимодействий

Санкт-Петербург

Researcher, Cell Interactions Laboratory, Department of Immunology and Cell Interactions

St. Petersburg

КостинН. А.KostinN. A.

сотрудник ресурсного центра «Развитие молекулярных и клеточных технологий»

Санкт-Петербург

Associate

St. Petersburg

МихайловаВ. А.MikhailovaV. A.

к.б.н., старший научный сотрудник лаборатории межклеточных взаимодействий, отдел иммунологии и межклеточных взаимодействий; старший преподаватель

Санкт-Петербург

PhD (Biology), Senior Research Associate, Cell Interactions Laboratory, Department of Immunology and Cell Interactions; Senior Lecturer

St. Petersburg

СельковС. А.SelkovS. A.

д.м.н., профессор, руководитель отдела иммунологии и межклеточных взаимодействий

Санкт-Петербург

PhD, MD (Medicine), Professor, Head, Immunology and Cell Interactions Department

St. Petersburg

СоколовД. И.SokolovD. I.

д.б.н., заведующий лабораторией межклеточных взаимодействий, отдел иммунологии и межклеточных взаимодействий; доцент

Санкт-Петербург

PhD, MD (Biology), Head, Cell Interactions Laboratory; Associate Professor

St. Petersburg

ФГБНУ «Научно-исследовательский институт акушерства, гинекологии и репродуктологии имени Д.О. Отта»РоссияD. Ott Research Institute of Obstetrics, Gynecology and ReproductologyRussian FederationРесурсный центр «Развитие молекулярных и клеточных технологий», Санкт-Петербургский государственный университетРоссияResource Center for Development of Molecular and Cell Technologies, St. Petersburg State UniversityRussian FederationФГБНУ «Научно-исследовательский институт акушерства, гинекологии и репродуктологии имени Д.О. Отта»; ГБОУ ВПО «Первый Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова» Министерства здравоохранения РФРоссияD. Ott Research Institute of Obstetrics, Gynecology and Reproductology; First St. Petersburg State I. Pavlov Medical UniversityRussian Federation201911072019213427440Copyright © Маркова К.Л., Степанова О.И., Шевелева А.Р., Костин Н.А., Михайлова В.А., Сельков С.А., Соколов Д.И., 20192019Маркова К.Л., Степанова О.И., Шевелева А.Р., Костин Н.А., Михайлова В.А., Сельков С.А., Соколов Д.И.Markova K.L., Stepanova O.I., Sheveleva A.R., Kostin N.A., Mikhailova V.A., Selkov S.A., Sokolov D.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.mimmun.ru/mimmun/article/view/1816

Регуляция ангиогенеза в зоне маточно-плацентарного контакта определяет адекватную инвазию трофобласта, формирование и развитие плаценты, успешное протекание беременности. Наиболее значительное влияние на ангиогенез оказывают NK-клетки, макрофаги, трофобласт. На сегодняшний день довольно подробно описаны функции клеток-участников формирования плаценты как по отдельности (in vitrо), так и в составе тканей (in situ). Однако до сих пор не создано моделей, отражающих взаимодействие NK-клеток, трофобласта и эндотелия в ходе ангиогенеза. До настоящего времени остается неразрешенным вопрос о вкладе каждой клеточной популяции в регуляцию не только ангиогенеза в плаценте, но и о перекрестной регуляции функций клеток-участников. Поэтому целью настоящего исследования явилось изучение контактного и дистантного влияния NK-клеток на образование капилляроподобных структур сокультурой эндотелиальных клеток и клеток трофобласта под влиянием различных цитокинов (bFGF, VЕGF, PlGF, TGF-β, IL-8, IFNγ, IL-1β). Введение в сокультуру ЭК и трофобласта NK-клеток в условиях дистантного и контактного культивирования не изменяло длину капилляроподобных структур, образованных ЭК. При контактном культивировании NK-клеток с сокультурой ЭК и трофобласта в присутствии IL-1β длина капилляроподобных структур не изменялась по сравнению с культивированием в тех же условиях, но в отсутствие цитокина. При дистантном культивировании NK-клеток с сокультурой ЭК и трофобласта в присутствии IL-1β произошло увеличение длины капилляроподобных структур по сравнению с культивированием в тех же условиях, но в отсутствие цитокина. При контактном, но не дистантном, культивировании NK-клеток с сокультурой ЭК и трофобласта в присутствии VЕGF длина капилляроподобных структур была больше по сравнению с культивированием в тех же условиях, но в отсутствие цитокина. В трех- компонентной клеточной системе провоспалительный цитокин IFNγ не оказывал эффекта в отношении ангиогенеза. При дистантном, но не контактном, культивировании NK-клеток с сокультурой ЭК и трофобласта в присутствии TGF-β длина капилляроподобных структур была меньше по сравнению с культивированием в тех же условиях, но в отсутствие цитокина. В условиях дистантного культивирования TGF-β запускает ингибирующий ангиогенез сигнал от NK- клеток. Установлено снижение длины капилляроподобных структур в условиях трехкомпонентной клеточной сокультуры в присутствии проангиогенных факторов: IL-8, PlGF (только при контактном культивировании) и bFGF (при контактном и дистантном культивировании). Таким образом, эффекты цитокинов в отношении ангиогенеза в трехкомпонентной сокультуре (NK-клетки, трофобласт, эндотелий) отличаются от установленных ранее в однокомпонентных (только эндотелий) и двухкомпонентных (сокультура эндотелия и трофобласта) клеточных моделях. Данные, полученные в настоящем исследовании, свидетельствуют о наличии в плаценте цитокиново-контактной регуляции межклеточных взаимодействий.

Regulation of angiogenesis in the utero-placental bed determines adequate trophoblast invasion, placenta formation and development, as well as successful course of pregnancy. Natural killer (NK) cells, macrophages and trophoblast have the most significant effect on angiogenesis. To date, the functions of cells participating in placenta formation have been described in detail, both individually (in vitrо) and in tissues (in situ). However, no models have yet been created that reflect the interactions of NK cells, trophoblast and endothelium during angiogenesis. It remains unclear, how each cell population contributes to placental angiogenesis regulation, and to the cross-regulation of participating cell functions. Therefore, the aim of this research was to study contact and distant effects of NK cells upon formation of tube-like structures through co-culture of endothelial and trophoblast cells influenced by various cytokines (bFGF, VEGF, PlGF, TGF-β, IL-8, IFNγ and IL-1β). Introduction of NK cells to the co-culture of endothelial and trophoblast cells under conditions of both contact and distance-dependent culturing did not change the length of tube-like structures formed by endothelial cells. During contact-dependent culturing of NK cells with co-culture of endothelial and trophoblast cells in presence of IL-1β, the length of tubule-like structures remained unchanged, compared with the length of tube-like structures formed under the same culturing conditions, but without the cytokine added. During distant culturing of NK cells with co-culture of endothelial and trophoblast cells in the presence of IL-1β, the length of tube-like structures increased as compared with those formed under the same culturing conditions but without the cytokine. During contact-dependent (but not distant) culturing of NK cells with the co-culture of endothelial and trophoblast cells in the presence of VEGF, the length of tube-like structures was greater than those formed under the same culturing conditions but without the cytokine. When used in a three-component cell system, the pro-inflammatory cytokine IFNγhad no effect upon angiogenesis. During distant (but not contact-dependent) culturing of NK cells with co-culture of endothelial and trophoblast cells in the presence of TGF-β, the length of tube-like structures was less than the length of tube-like structures formed under the same culturing conditions but without the cytokine. Under conditions of distant culturing, TGF-βtriggered a signal in NK cells that inhibited angiogenesis. Decreased length of tube-like structures under conditions of a three-component cell co-culture in the presence of the following pro-angiogenic factors was revealed: IL-8, PlGF (during contact-dependent culturing only) and bFGF (during both contact-dependent and distant culturing). Thus, the effects of cytokines upon angiogenesis in a three-component co-culture (NK cells, trophoblast and endothelium) differed from those revealed previously in single-component (endothelium only) and two-component (co-culture of endothelium and trophoblast) cell models. The results of these experiments indicated that regulation of placental cell interactions involved both cellular contacts and effects produced by cytokines.

эндотелиальные клеткитрофобластNK-клеткиангиогенезцитокиныendothelial cellstrophoblastnatural killer cellsangiogenesiscytokinesThis research was supported by the Russian Science Foundation (grant No. 17-15-01230: culturing of endothelial cells, assessment of the formation of tube-like structures) and partially carried out within the framework of the government program No. ААААА 18-118011020016-9: culturing of NK cells and trophoblast cells. The confocal microscopy was carried out using the equipment of the Resource Center for the Molecular and Cell Technologies Development under St. Petersburg State University, St. Petersburg, Russia.ReferencesАйламазян Э.К., Степанова О.И., Сельков С.А., Соколов Д.И. Клетки иммунной системы матери и клетки трофобласта: «Конструктивное сотрудничество» ради достижения совместной цели // Вестник Российской академии медицинских наук, 2013. Т. 11. С. 12-21.Ailamazian E.K., Stepanova O.I., Selkov S.A., Sokolov D.I. 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The authors declare that there are no conflicts of interest present.