mimmunМедицинская иммунологияMedical Immunology (Russia)1563-06252313-741XSPb RAACI10.15789/1563-0625-2016-4-339-346mimmun-1056Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESИЗМЕНЕНИЯ УРОВНЯ ЭКСПРЕССИИ ГЕНОВ Glut1, mTOR И AMPK1α ЛИМФОЦИТАМИ ПАНКРЕАТИЧЕСКИХ ЛИМФАТИЧЕСКИХ УЗЛОВ КРЫС ПРИ ЭКСПЕРИМЕНТАЛЬНОМ САХАРНОМ ДИАБЕТЕСHANGES OF Glut1, mTOR AND AMPK1α GENE EXPRESSION IN PANCREATIC LYMPH NODE LYMPHOCYTES OF RATS WITH EXPERIMENTAL DIABETES MELLITUSПутилинД. А.PutilinD. A.

ассистент кафедры нормальной физиологии,

69035, г. Запорожье, пр. Маяковского, 26

Assistant Professor, Department of Normal Physiology, 

69035, Zaporozhye, Mayakovsky ave, 26

alexkamyshny@yandex.ruКамышныйА. М.KamyshnyiA. M.

д.м.н., профессор, заведующий кафедрой микробиологии, вирусологии и иммунологии, заведующий молекулярно-генетической лабораторией

PhD, MD (Medicine), Professor, Chief, Department of Microbiology, Virology, and Immunology, Head, Laboratory of Molecular Genetics

Запорожский государственный медицинский университетУкраинаZaporozhye State Medical UniversityUkraine201622082016184339346Copyright © Путилин Д.А., Камышный А.М., 20162016Путилин Д.А., Камышный А.М.Putilin D.A., Kamyshnyi A.M.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.mimmun.ru/mimmun/article/view/1056

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 исследуемых 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 моноклональных антител. Установлено, что гипергликемия вызывала транскрипционную индукцию генов 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 неделе 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.

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 Systems, Bio-Rad, USA). Relative gene expression levels were calculated as ratios to GAPDH reference gene using ΔΔCt method. Statistical analysis was performed with available “Bio-Rad СFX Manager 3.1” software (Bio-Rad, USA). The mTOR+ positive lymphocytes were identified by means of monoclonal antibodies, using an indirect immunofluorescence method. Hyperglycemia was accompanied by transcriptional induction of glucose transporter Glut1 gene (9.9 to 28.9-fold, p < 0.05), and mTOR protein kinase gene (5.3 to 3.3-fold, p < 0.05) in PLN. Development of diabetes was also associated with increase by 24-34% in total mTOR+ cell numbers in PLN at the 5th week of developing diabetes (p <  0.05) and increased concentrations of rapamycin target in the immunopositive cells. Metformin administration to diabetic rats was followed by increased AMPK1α mRNA level of by 87% (p < 0,05) at the 3rd week, and 38-fold (p < 0,05), at the 5th week of SIDM development and inhibition of mTOR expression in PLN (3 to 14.7-fold, p < 0.05) accompanied by a 40 per cent decrease (p < 0.05) in total density of mTOR+ cells in PLN lymph cords of the rats following 5 weeks of SIDM.

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