rspНаучно-практическая ревматологияRheumatology Science and Practice1995-44841995-4492IMA-PRESS, LLC10.14412/1995-4484-2012-1111rsp-1031Research ArticleСтатьиArticlesПовышенная экспрессия регуляторных генов в крови и суставном хряще больных ревматоидным артритомEnhanced regulatory gene expressions in the blood and articular cartilage of patients with rheumatoid arthritisChetinaElena VasilyevnaChetinaElena Vasilyevnaetchetina@mail.ruSemyonovaL ASemyonovaL A-ЛогуновА. Л.LogunovA L-КоломацкийВ. В.KolomatskyV V-МакаровМ. А.MakarovM A-РоскидайлоА. А.RoskidailoA A-NaryshkinE ANaryshkinE A-МакаровС. А.MakarovS A-201215082012504№4 (2012)4448Copyright © Chetina E.V., Semyonova L.A., Логунов А.Л., Коломацкий В.В., Макаров М.А., Роскидайло А.А., Naryshkin E.A., Макаров С.А., 20122012Chetina E.V., Semyonova L.A., Логунов А.Л., Коломацкий В.В., Макаров М.А., Роскидайло А.А., Naryshkin E.A., Макаров С.А.Chetina E.V., Semyonova L.A., Logunov A.L., Kolomatsky V.V., Makarov M.A., Roskidailo A.A., Naryshkin E.A., Makarov S.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://rsp.mediar-press.net/rsp/article/view/1031

Цель - исследовать соотношение экспрессии нетканеспецифических регуляторных генов mTOR, р21, ATG1, каспазы 3, фактора некроза опухолей (ФНО а) и интерлейкина 6 (ИЛ 6), а также генов матриксной металлопротеиназы 13 (ММП 13) и коллагена Х типа (КОЛ10А1), ассоциированных с резорбцией хряща ММП 13 и КОЛ10А1 в крови и хряще коленных суставов больных ревматоидным артритом (РА). Материал и методы. Исследовано 25 образцов суставного хряща дистальных мыщелков бедренной кости 15 больных РА (средний возраст 52,4±9,1 года), которым проведено эндопротезирование коленных суставов, и 10 здоровых людей (средний возраст 36,0±9,1 года), включенных в контрольную группу. Кроме того, проанализированы 28 образцов крови больных РА, взятой перед эндопротезированием коленных суставов (средний возраст 52+7,6 года), и 27 образцов крови здоровых людей (контроль, средний возраст 53,6±8,3 года). Посредством количественной полимеразной цепной реакции в режиме реального времени в образцах крови и хряща определены уровни экспрессии генов mTOR, р21, ATG1, каспазы 3, ФНО а, ИЛ 6, КОЛ10A1 и ММП 13. Содержание белкового эквивалента исследуемых генов р70-S6K (активируемого mTOR), р21 и каспазы 3 определяли в лизатах изолированных лимфоцитов с использованием готовых наборов для иммуноферментного анализа (ELISA). Суммарный белок в клеточных экстрактах определяли методом Бредфорда. Результаты. Экспрессия генов mTOR, ATG1, p21, ФНО а, MMП13 и КОЛ10A1 в образцах хряща больных РА на поздней стадии заболевания была значительно выше, чем у здоровых людей. В то же время экспрессия ИЛ 6 оказалась значительно ниже, чем в контрольной группе. В крови больных РА экспрессия генов mTOR, ATG1, p21, ФНО а и ИЛ 6 значительно превышала уровни их экспрессии у доноров. Уровни экспрессии каспазы 3 существенно не различались в крови больных РА и здоровых людей. Экспрессия MMП13 и KQn10A1 α в крови не обнаружена. Высокая экспрессия генов mTOR, p21 и каспазы 3 в крови сопровождалась повышенной концентрацией соответствующих белков в лизатах клеток больных РА по сравнению со здоровыми лицами. Заключение. Полученные результаты впервые свидетельствуют о повышенной экспрессии регуляторных генов mTOR, ATG1, p21 и ФНО а в крови и суставном хряще больных РА. Эти изменения сопровождаются повышенной экспрессией гена ММП 13, ответственного за резорбцию хряща. Поэтому повышение экспрессии исследованных регуляторных генов в крови больных РА может свидетельствовать о деструкции суставного хряща.

Objective: to study the expression ratio of the non-tissue specific regulatory genes mTOR, р21, ATG1, caspase 3, tumor necrosis factor-а (TNF-а), and interleukin-6 (IL-6), as well as matrix metalloproteinase 13 (MMP-13) and X type collagen (COL10A1), cartilage resorption-associated MMP13 and COL10A1 in the blood and knee articular cartilage in patients with rheumatoid arthritis (RA). Subjects and methods. Twenty-five specimens of the distal femoral articular cartilage condyles were studied in 15 RA patients (mean age 52.4+9.1 years) after endoprosthetic knee joint replacement and in 10 healthy individuals (mean age 36.0+9.1 years) included into the control group. Twenty-eight blood samples taken from 28 RA patients (aged 52+7.6 years) prior to endoprosthetic knee joint replacement and 27 blood samples from healthy individuals (mean age 53.6+8.3 years; a control group) were also analyzed. Real-time quantitative polymerase chain reaction was applied to estimate the expression of the mTOR, p21, ATG1, caspase 3, TNF-а, IL- 6, COL0A1, and MMP-13 genes. The levels of a protein equivalent in the p70-S6K(activated by mTOR), p21, and caspase 3 genes concerned was measured in the isolated lymphocyte lysates, by applying the commercially available ELISA kits. Total protein in the cell extracts was determined using the Bradford assay procedure. Results. The cartilage samples from patients with end-stage RA exhibited a significantly higher mTOR, ATG1, p21, TNFа, MMP-13, and COL10A1 gene expressions than did those from the healthy individuals. At the same time, IL6 gene expression was much lower than that in the control group. The expressions of the mTOR, ATG1, p21, TNFа, and IL 6 genes in the blood of RA patients were much greater than those in the donors. Caspase 3 expression did not differ essentially in the bloods of the patients with RA and healthy individuals. The bloods failed to show MMP-13 and COL10A1 expressions. High mTOR and p21 gene expressions were accompanied by the elevated concentrations of the corresponding proteins in the cell lysates of the patients with RA compared to the controls. Conclusion. The findings suggest for the first time that regulatory mTOR, ATG1, p21, and TNFа gene expressions are enhanced in the blood and articular cartilage of RA patients. These changes are accompanied by the increased expression of MMP 13 gene that is responsible for articular cartilage resorption. Therefore, the higher expression of the examined regulatory genes in the blood of RA patients may be indicative of articular cartilage degradation.

ревматоидный артритэкспрессия геновкровьсуставной хрящmTORrheumatoid arthritisgene expressionmTORbloodarticular cartilageReferences<div><p>Firenstein G.S. Evolving concepts of rheumatoid arthritis. Nature 2003;423:356-61.</p><p>Rindfleisch J.A., Muller D. Diagnosis and management of rheumatoid arthritis. Am Fam Physician 2005;72:1037-47.</p><p>Karouzakis E., Neidhart M., Gay R.E., Gay S. Molecular and cellular basis of rheumatoid joint destruction. Immunology Letters 2006;106:8-13.</p><p>Hay N., Sonnenberg N. Upstream and downstream of mTOR. Genes Dev 2004;18:1926-45.</p><p>Djavahery-Mergny M., Amelotti M., Mathieu J. et al. NF-kB activation represses tumor necrosis factor-a-induced autophagy. J Biol Chem 2006;281:30373-82.</p><p>Laragione T., Gulko P.S. mTOR regulates the invasive properties of synovial fibroblasts in rheumatoid arthritis. Mol Med 2010;16:352-8</p><p>Haupl T., Ostensen M., Grützkau A. et al. Reactivation of rheumatoid arthritis after pregnancy: increased phagocyte and recurring lymphocyte gene activity. Arthr Rheum 2008;58:2981-92.</p><p>Tchetina E.V., Demidova N.V., Semyenova L.A. et al. Differential expression of mammalian target of rapamycin (mTOR) in the peripheral blood of early-stage rheumatoid arthritis patients as a prognostic marker of the disease activity and knee joint destruction: a two year follow up study. Ann Rheum Dis 2010;69(Suppl.3):675.</p><p>Carlson R.P., Hartman D.A., Tomchek L.A. et al. Rapamycin, a potential disease-modifying antiarthritic drug. J Pharmacol Exp Ther 1993;266:1125-38.</p><p>Čejka D., Hayer S., Niederreiter B. et al. Mammalian target of rapamycin signaling is crucial for joint destruction in experimental arthritis and is activated in osteoclasts from patients with rheumatoid arthritis. Arthr Rheum 2010;62:2294-302</p><p>Bruyn G.A., Tate G., Caeiro F. et al. Everolimus in patients with rheumatoid arthritis receiving concomitant methotrexate: a 3-month, double-blind, randomised, placebo-controlled, parallel-group, proof-of-concept study. Ann Rheum Dis 2008;67:1090-5.</p><p>Lum J.J., DeBerardinis R.J., Thompson C.B. Autophagy in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol 2005;6:439-48.</p><p>Lleo A., Invernizzi P., Selmi C. et al. Autophagy: highlighning a novel player in the autoimmunity scenario. J Autoimmun 2007;2:61-8.</p><p>Lu B., Capan E., Li C. Autophagy induction and autophagic cell death in effector T cells. Autophagy 2007;3:158-9.</p><p>Eguchi K. Apoptosis in autoimmune diseases. Intern Med 2001;40:275-84.</p><p>Catrina A.I., Ulfgren A.K., Lindblad S. et al. Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium. Ann Rheum Dis 2002;61:934-6.</p><p>Rasa K., Scheel-Toellner D., Lee C.-Y. et al. Synovial fluid apoptosis is inhibited in patients with very early rheumatoid arthritis. Arthr Res Ther 2006;8:R120.</p><p>Shin Y.J., Han S.H., Kim D.S. et al. Autophagy induction and CHOP under-expression promotes survival of fibroblasts from rheumatoid arthritis patients under endoplasmic reticulum stress. Arthr Res Ther 2010;12:R19.</p><p>Wood K., Twigg H.L., Doseff A.I. Dysregulation of CD8+ lymphocyte apoptosis, chronic disease, and immune regulation. Front Biosci 2009;14, 3771-81.</p><p>Grcevic D., Jajic Z., Kovacic N. et al. Peripheral blood expression profiles of bone morphogenetic proteins, tumor necrosis factor-superfamily molecules, and transcription factor Runx2 could be used as markers of the form of arthritis, disease activity, and therapeutic responsiveness. J Rheumatol 2010;37:246-56.</p><p>Mahr S., Burmester G.R., Hilke D. et al. Cis- and trans-acting gene regulation is associated with osteoarthritis. Am J Hum Genet 2006;78:793-803.</p><p>Лимфоциты - методы. Под ред. Д. Клауса. М.:Мир,1990;52-3.</p><p>Kruger N.J. The Bradford method for protein quantitation. Methods Mol Biol 1994;32:9-15.</p><p>Четина Е.В., Ди Батиста Д., Пул А.Р. Роль простагландина E2 в ингибировании разрушения коллагена суставного хряща больных остеоартрозом. Науч-практич ревматол 2009;3:18-23.</p><p>Tchetina E.V., Squires G., Poole A.R. Increased type II collagen degradation and very early focal cartilage degeneration is associated with upregulation of chondrocyte differentiation related genes in early human articular cartilage lesions. J Rheumatol 2005;32:876-86.</p><p>Четина Е.В., Пул А.Р. Роль ростовых факторов в подавлении разрушения коллагена и дифференциации хондроцитов при остеоартрозе. Bестн РАМН 2008;5:15-21.</p><p>Gartel A.L., Tyner A.L. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther 2002;1:639-49.</p><p>Peng S.L. Fas (CD95)-related apoptosis and rheumatoid arthritis. Rheumatology (Oxford) 2006;45:26-30.</p><p>Raghav S.K., Gupta B., Agrawal C. et al. Expression of TNF-alpha and related signaling molecules in the peripheral blood mononuclear cells of rheumatoid arthritis patients. Mediators Inflamm 2006;3:12682.</p><p>Dodds R.A., Merry K., Littlewood A., Gowen M. Expression of mRNA for IL1 beta, IL6 and TGF beta 1 in developing human bone and cartilage. J Histochem Cytochem 1994;42:733-44.</p><p>Blagosklonny M.V. Cell senescence and hypermitogenic arrest. EMBO Report 2003;4:358-62.</p><p>Demidenko Z.N., Blagosklonny M.V. Growth stimulation leads to cellular senescence when the cell cycle is blocked. Cell Cycle 2008;7:3355-61.</p><p>Четина Е.В. Механизмы эмбриогенеза при остеоартрозе: роль дифференцировки хондроцитов в резорбции суставного хряща. Науч-практич ревматол 2010;3:65-77.</p></div><br /><div><p>Firenstein G.S. Evolving concepts of rheumatoid arthritis. Nature 2003;423:356-61.</p><p>Rindfleisch J.A., Muller D. Diagnosis and management of rheumatoid arthritis. Am Fam Physician 2005;72:1037-47.</p><p>Karouzakis E., Neidhart M., Gay R.E., Gay S. Molecular and cellular basis of rheumatoid joint destruction. Immunology Letters 2006;106:8-13.</p><p>Hay N., Sonnenberg N. Upstream and downstream of mTOR. Genes Dev 2004;18:1926-45.</p><p>Djavahery-Mergny M., Amelotti M., Mathieu J. et al. NF-kB activation represses tumor necrosis factor-a-induced autophagy. J Biol Chem 2006;281:30373-82.</p><p>Laragione T., Gulko P.S. mTOR regulates the invasive properties of synovial fibroblasts in rheumatoid arthritis. Mol Med 2010;16:352-8</p><p>Haupl T., Ostensen M., Grützkau A. et al. Reactivation of rheumatoid arthritis after pregnancy: increased phagocyte and recurring lymphocyte gene activity. Arthr Rheum 2008;58:2981-92.</p><p>Tchetina E.V., Demidova N.V., Semyenova L.A. et al. Differential expression of mammalian target of rapamycin (mTOR) in the peripheral blood of early-stage rheumatoid arthritis patients as a prognostic marker of the disease activity and knee joint destruction: a two year follow up study. Ann Rheum Dis 2010;69(Suppl.3):675.</p><p>Carlson R.P., Hartman D.A., Tomchek L.A. et al. Rapamycin, a potential disease-modifying antiarthritic drug. J Pharmacol Exp Ther 1993;266:1125-38.</p><p>Čejka D., Hayer S., Niederreiter B. et al. Mammalian target of rapamycin signaling is crucial for joint destruction in experimental arthritis and is activated in osteoclasts from patients with rheumatoid arthritis. Arthr Rheum 2010;62:2294-302</p><p>Bruyn G.A., Tate G., Caeiro F. et al. Everolimus in patients with rheumatoid arthritis receiving concomitant methotrexate: a 3-month, double-blind, randomised, placebo-controlled, parallel-group, proof-of-concept study. Ann Rheum Dis 2008;67:1090-5.</p><p>Lum J.J., DeBerardinis R.J., Thompson C.B. Autophagy in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol 2005;6:439-48.</p><p>Lleo A., Invernizzi P., Selmi C. et al. Autophagy: highlighning a novel player in the autoimmunity scenario. J Autoimmun 2007;2:61-8.</p><p>Lu B., Capan E., Li C. Autophagy induction and autophagic cell death in effector T cells. Autophagy 2007;3:158-9.</p><p>Eguchi K. Apoptosis in autoimmune diseases. Intern Med 2001;40:275-84.</p><p>Catrina A.I., Ulfgren A.K., Lindblad S. et al. Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium. Ann Rheum Dis 2002;61:934-6.</p><p>Rasa K., Scheel-Toellner D., Lee C.-Y. et al. Synovial fluid apoptosis is inhibited in patients with very early rheumatoid arthritis. Arthr Res Ther 2006;8:R120.</p><p>Shin Y.J., Han S.H., Kim D.S. et al. Autophagy induction and CHOP under-expression promotes survival of fibroblasts from rheumatoid arthritis patients under endoplasmic reticulum stress. Arthr Res Ther 2010;12:R19.</p><p>Wood K., Twigg H.L., Doseff A.I. Dysregulation of CD8+ lymphocyte apoptosis, chronic disease, and immune regulation. Front Biosci 2009;14, 3771-81.</p><p>Grcevic D., Jajic Z., Kovacic N. et al. Peripheral blood expression profiles of bone morphogenetic proteins, tumor necrosis factor-superfamily molecules, and transcription factor Runx2 could be used as markers of the form of arthritis, disease activity, and therapeutic responsiveness. J Rheumatol 2010;37:246-56.</p><p>Mahr S., Burmester G.R., Hilke D. et al. Cis- and trans-acting gene regulation is associated with osteoarthritis. Am J Hum Genet 2006;78:793-803.</p><p>Лимфоциты - методы. Под ред. Д. Клауса. М.:Мир,1990;52-3.</p><p>Kruger N.J. The Bradford method for protein quantitation. Methods Mol Biol 1994;32:9-15.</p><p>Четина Е.В., Ди Батиста Д., Пул А.Р. Роль простагландина E2 в ингибировании разрушения коллагена суставного хряща больных остеоартрозом. Науч-практич ревматол 2009;3:18-23.</p><p>Tchetina E.V., Squires G., Poole A.R. Increased type II collagen degradation and very early focal cartilage degeneration is associated with upregulation of chondrocyte differentiation related genes in early human articular cartilage lesions. J Rheumatol 2005;32:876-86.</p><p>Четина Е.В., Пул А.Р. Роль ростовых факторов в подавлении разрушения коллагена и дифференциации хондроцитов при остеоартрозе. Bестн РАМН 2008;5:15-21.</p><p>Gartel A.L., Tyner A.L. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther 2002;1:639-49.</p><p>Peng S.L. Fas (CD95)-related apoptosis and rheumatoid arthritis. Rheumatology (Oxford) 2006;45:26-30.</p><p>Raghav S.K., Gupta B., Agrawal C. et al. Expression of TNF-alpha and related signaling molecules in the peripheral blood mononuclear cells of rheumatoid arthritis patients. Mediators Inflamm 2006;3:12682.</p><p>Dodds R.A., Merry K., Littlewood A., Gowen M. Expression of mRNA for IL1 beta, IL6 and TGF beta 1 in developing human bone and cartilage. J Histochem Cytochem 1994;42:733-44.</p><p>Blagosklonny M.V. Cell senescence and hypermitogenic arrest. EMBO Report 2003;4:358-62.</p><p>Demidenko Z.N., Blagosklonny M.V. Growth stimulation leads to cellular senescence when the cell cycle is blocked. Cell Cycle 2008;7:3355-61.</p><p>Четина Е.В. Механизмы эмбриогенеза при остеоартрозе: роль дифференцировки хондроцитов в резорбции суставного хряща. Науч-практич ревматол 2010;3:65-77.</p></div><br />

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