vestimИзвестия Национальной академии наук Беларуси. Серия медицинских наукProceedings of the National Academy of Sciences of Belarus, Medical series1814-60232524-2350The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1814-6023-2020-17-3-263-274vestim-687Research ArticleКЛИНИЧЕСКАЯ И ЭКСПЕРИМЕНТАЛЬНАЯ МЕДИЦИНАCLINICAL AND EXPERIMENTAL MEDICINEВлияние иммунных факторов пациентов с сахарным диабетом на жизнеспособность клеток кожи in vitroImpact of diabetic (diabetes mellitus) patients immune factors on the skin cell viability in vitroМашковаМ. А.MashkovaM. A.

Машкова Мария Александровна – аспирант, ассистент

пр-т Дзержинского, 83, 220116, г. Минск

Maryia А. Mashkova – Postgraduate student, Assistant

83, Dzerzhynskii Ave., 220116, Minsk

mashkova.maryia@gmail.comМохортТ. В.MokhortT. V.

Мохорт Татьяна Вячеславовна – доктор медицинских наук, профессор, заведующий кафедрой

пр-т Дзержинского, 83, 220116, г. Минск

Tatiana V. Mokhort – D. Sc. (Med.), Professor, Head of the Department

83, Dzerzhynskii Ave., 220116, Minsk

tatsianamokhort@gmail.comГорановВ. А.GoranovV. A.

Горанов Виталий Анатольевич – кандидат медицинских наук, научный сотрудник

пр-т Дзержинского, 83, 220116, г. Минск

Vitaly A. Goranov – Ph. D. (Med.), Researcher

83, Dzerzhynskii Ave., 220116, Minsk

vitgoranov@gmail.comБелорусский государственный медицинский университетBelarusian State Medical University202029082020173263274Copyright © Машкова М.А., Мохорт Т.В., Горанов В.А., 20202020Машкова М.А., Мохорт Т.В., Горанов В.А.Mashkova M.A., Mokhort T.V., Goranov V.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestimed.belnauka.by/jour/article/view/687

На сегодняшний день большой научный интерес представляет изучение роли иммунных факторов в патогенезе язвы стопы при сахарном диабете (СД). Цель исследования – изучить с помощью 3D-клеточной модели in vitro характер взаимодействия между клетками кожи (кератиноциты и фибробласты) и факторами иммунной системы, выделенными из периферической крови пациентов с СД второго типа (СД2) и лиц с СД2 с хронической язвой стопы. Для создания 3D-клеточной модели кожи использовали первичную культуру фибробластов, кератиноциты линии HaCaT (иммортализованные кератиноциты), сыворотку крови и мононуклеары периферической крови пациентов – 9 здоровых доноров без СД, 9 пациентов с СД2 и 9 пациентов с СД2 с синдромом диабетической стопы (СДС) с хронической язвой. При сокультивировании клеток кожи с мононуклеарной суспензией здоровых доноров, пациентов с СД2 и лиц с СДС доля жизнеспособных фибробластов составила 95,40 [91,75; 99,05] % vs 83,78 [79,03; 89,53] % vs 70,18 [66,38; 72,10] % (H = 21,259, p < 0,001), кератиноцитов – 96,40 [92,82; 100,50] % vs 93,61 [86,80; 97,10] % vs 92,87 [85,15; 95,25] % (H = 4,459, p = 0,108) соответственно. При отдельном использовании 20 %-ной сыворотки крови доля жизнеспособных фибробластов в этих группах составила 95,61 [92,39; 100,19] % vs 95,80 [88,99; 102,15] % vs 96,20 [99,69; 88,70] % (H  =  0,353, p  =  0,838), кератиноцитов – 99,40 [95,35; 102,05]  % vs 98,60 [90,55; 100,40]  % vs 94,79 [91,65; 98,16]  % (H = 3,030, p = 0,220) соответственно. Установлено, что мононуклеары, выделенные из периферической крови пациентов с СД2 и СД2 с СДС, значительно снижают выживаемость фибробластов при сокультивировании в 3D-модели кожи in vitro, не оказывая при этом достоверного влияния на жизнеспособность/выживаемость кератиноцитов. В то же время иммунные факторы, содержащиеся в сыворотке крови пациентов указанных выше групп, не оказывали достоверного эффекта на жизнеспособность клеток кожи в 3D-системе in vitro.

At the moment, a lot of scientific research focused on the role of immune mechanisms in diabetic foot ulcers development and impaired healing. A 3D skin culture system as a relevant skin model may prove valuable in investigating these mechanisms and may be a useful tool to study interactions between different cell types such as keratinocytes, fibroblasts, and immune cells. The aim of our research was to study keratinocytes and fibroblasts viability in co-culture with immune factors of patients with diabetes mellitus type 2 (DM2) and patients with diabetes and chronic foot ulcers in a 3D skin culture system. In this study, the multilayer 3D immunocompetent model of human skin comprising keratinocytes, fibroblasts, and mononuclears in an agarose-fibronectin gel was used. The human immortalized keratinocyte cell line, HaCaT, and primary fibroblast cell culture isolated from skin samples of healthy man in abdominal surgery were used for the 3D system. For the experiment 20 % serum of 9 patients with chronic diabetic foot ulcers (without active inflammation signs), 9 diabetic type 2 patients and 9 healthy people, and mononuclears of the same groups of patients were used. 9 experimental series with 3 repeats were carried out. Mononuclears of patients with DM2 and DM2 and diabetic foot syndrome (DFS) had a greater inhibitory effect on fibroblasts, significantly inhibiting their proliferation to a level of 83.78 [79.03; 89.53] % vs 70.18 [66.38; 72.10] % vs 95.40 [91.75; 99.05] %, H = 21.259, p <0.001 – DM2, DFS, and the control group, respectively. There was no significant difference in the cytoinhibitory effect of mononuclears on keratinocytes between different groups: 96.40 [92.82; 100.50] % vs 93.61 [86.80; 97.10] % vs 92.87 [85.15; 95.25] %, H = 4.459, p = 0.108 – control, DM2 and DFS group, respectively. Adding serum to the culture system influenced significantly the viability of neither keratynocytes – 99.40 [95.35; 102.05]  % vs 98.60 [90.55; 100.40] % vs 94.79 [91.65; 98.16] %, H = 3.030, p = 0.220 nor of fibroblasts – 95.61 [92.39; 100.19] % vs 95.80 [88.99; 102.15] % vs 96.20 [99.69; 88.70] %, H = 0.353, p = 0.838, control, DM2 and DFS group, respectively. It was determined that the fibroblasts vialability significantly decreases after introducing mononuclears of patients with DM and patients with DM and chronic diabetic foot ulcers to the co-culture system. Adding serum of these patient groups to the culture system doesn’t influence significantly the viability of skin cells.

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