ИЗМЕНЕНИЯ УРОВНЯ ЭКСПРЕССИИ ГЕНОВ Glut1, mTOR И AMPK1α ЛИМФОЦИТАМИ ПАНКРЕАТИЧЕСКИХ ЛИМФАТИЧЕСКИХ УЗЛОВ КРЫС ПРИ ЭКСПЕРИМЕНТАЛЬНОМ САХАРНОМ ДИАБЕТЕ Путилин Д.А., Камышный А.М. Запорожский государственный медицинский университет, г. Запорожье, УкраинаРезюме. С помощью молекулярно-генетических методов исследовали уровень экпрессии мРНК генов Glut1, mTOR и AMPK1α в ПЛУ крыс с экспериментальным стрептозотоцин-индуцированным сахарным диабетом (ЭСИСД) и после введений метформина. Для определения уровня мРНК иссле-дуемых генов проводили ОТ-ПЦР в реальном времени на амплификаторе CFX96™ Real-Time PCR Detection Systems ("Bio-Rad Laboratories, Inc.", США). Иммунопозитивные mTOR + лимфоциты были идентифицированы с помощью метода непрямой иммунофлюоресценции с использованием моно-клональных антител. Установлено, что гипергликемия вызывала транскрипционную индукцию ге-нов транспортеров глюкозы Glut1 (в 9,9-28,9 раз, p < 0,05) и протеинкиназы mTOR (в 5,3-3,3 раза, p < 0,05) в клетках ПЛУ. Развитие диабета также сопровождалось увеличением общего числа mTOR + клеток в ПЛУ на 5 неделе патологического процесса на 24-34% (р < 0,05) и концентрации мишени рапамицина в иммунопозитивных клетках. Введения метформина диабетическим крысам приводили к увеличению уровня мРНК гена AMPK1α на 87% (p < 0,05) на 3 неделе и в 38 раз (p < 0,05) на 5 не-деле развития ЭСИСД и угнетению экспрессии mTOR в ПЛУ (в 3-14,7 раз, p < 0,05), сопровождаясь снижением на 40% (р < 0,05) суммарной плотности mTOR + клеток в мякотных тяжах ПЛУ у животных с 5-ти недельным ЭСИСД. Ключевые слова: стрептозотоцин-индуцированный сахарный диабет, панкреатические лимфатические узлы, Glut1, mTOR, AMPK1α, экспрессия генов СHANGES OF Glut1, mTOR AND AMPK1α GENE EXPRESSION IN PANCREATIC LYMPH NODE LYMPHOCYTES OF RATS WITH EXPERIMENTAL DIABETES MELLITUSPutilin D.A., Kamyshnyi A.M. Zaporozhye State Medical University, Zaporozhye, UkraineAbstract. With the help of molecular genetic method we have investigated the level of mRNA gene expressions Glut1, mTOR and AMPK1α in PLN in pancreatic lymph nodes (PLN) of rats with streptozotocininduced diabetes mellitus (SIDM) and after administration of metformin. The levels of Glut1, mTOR and AMPK1α mRNA were determined by means of RT-PCR using CFX96™ thermocycler (Real-Time PCR Detection Адрес для переписки: Камышный Александр Михайлович Запорожский государственный медицинский университет 69035, Украина, г. Запорожье, пр. Маяковского, 26.

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Background: Peripheral tolerance can be mediated by extrathymic Aire-expressing cells (eTACs) and lymph node stromal cells (LNSCs) have recently been shown to induce T-cell tolerance by ectopically expressing and presenting range of peripheral tissue antigens (PTAs). New evidence shows that all types of LNSCs-including fibroblastic reticular cells, follicular DCs, and lymphatic endothelial cells-express PTAs. Ectopic expression of genes encoding PTAs in lymph nodes (LN) controlled by transcriptional regulators Aire and deformed autoregulatory factor 1 (Deaf1). Therefore, the aim of our study was to determine the effect of the levels of Deaf1 and Aire mRNA expression on the nature of Foxp3 + Treg cells differentiation during experimental STZ-induced diabetes mellitus (EDM) in rats pancreatic lymph nodes (PLN). Methods. To determine the level of mRNA Deaf1 and Aire expression was performed RT-PCR in real-time by thermocycler CFX96™ Real-Time PCR Detection Systems. The relative level of gene expression were studied with rat reference genes GAPDH by the method ΔΔ Ct. Statistical analysis were conducted using available software «Bio-Rad СFX Manager 3.1». The Foxp3 +immunopositive lymphocytes were determined using an indirect immunofluorescence technique with using a monoclonal rat antibody. Results: Development of EDM was accompanied by decreased the expression levels of the transcriptional regulator Deaf1 and Aire in rats PLN. So, Deaf1 expression is decreased 4,2-fold in rats PLN with 3-week EDM and 2,5fold in rats with 5-week EDM. Aire expression is decreased 2-fold in rats PLN with 3-week EDM and 50-fold in rats with 5-week EDM. Reduced Deaf1 and Aire mRNA expression during EDM associated with an decreased of total amount of Treg in the PLN, led to changes of distribution into individual classes of FoxP3 + lymphocytes and FoxP3 concentration in immunopositive cells. Conclusions: development of EDM due to a breakdown in peripheral immune tolerance. This contributes to progression of diabetes mellitus.

Type 1 diabetes mellitus is a T-cell mediated autoimmune disease characterized by the destruction of β-cells of the pancreas. Numerous studies have demonstrated the key role of FoxP3 + regulatory T-cells in the development of type 1 diabetes. The aim of our study:1) to determine the expression patterns of transcription factor FoxP3 in the pancreatic lymph node cells in animal model of diabetes mellitus and 2) to assay an effect of metformin on these processes. Methods and results.The study was conducted on 60 male Wistar rats with streptozotocin-induced modeling diabetes with various duration of the diabetes. Within development of type 1 diabetes mellitus the number of Treg pancreatic lymph nodes has been reduced. Conclusion.These fi ndings demonstrate the ability of metformin to increase the number of Treg.

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