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GRNTI 76.03 Медико-биологические дисциплины
GRNTI 76.33 Гигиена и эпидемиология
OKSO 14.04.02 Ядерные физика и технологии
OKSO 31.06.2001 Клиническая медицина
OKSO 31.08.08 Радиология
OKSO 32.08.12 Эпидемиология
BBK 51 Социальная гигиена и организация здравоохранения. Гигиена. Эпидемиология
BBK 534 Общая диагностика
TBK 5708 Гигиена и санитария. Эпидемиология. Медицинская экология
TBK 5712 Медицинская биология. Гистология
TBK 5734 Медицинская радиология и рентгенология
TBK 6212 Радиоактивные элементы и изотопы. Радиохимия
Purpose: To conduct a comparative assessment of human mesenchymal stem cells (MSCs) exposed to ultrahigh doses of bremsstrahlung photon radiation at liquid nitrogen temperature (–196 °C) and room temperature (+22 °C) on the yield of residual DNA double-strand breaks (DSBs) and proliferative activity of thawed MSCs. Material and methods: Isolation and cultivation of MSCs was carried out according to standard methods. Dimethyl sulfoxide (DMSO) at a final concentration of 10 % was used for cells cryopreservation. The cells were irradiated with bremsstrahlung photon radiation with photon nominal energy 5 MeV, using the UELR-10-100-T-100 accelerator (Russia). Cells were irradiated at the doses of 50 and 500 Gy at a temperature of +22 °C and –196 °C. The immunocytochemical analysis of γH2AX foci (marker of DNA DSBs) was used for the assessment of the yield of residual DNA DSBs. The number of Ki67-positive cells (protein marker of cell proliferation) was analyzed for assessment of the cell proliferative activity. Results: The results showed that48 hours after irradiation of MSCs at a dose of 50 Gy the number of residual γH2AX foci in the nuclei of MSCs irradiated at +22 °C was about 3.2 times (p = 0.0002) higher than in those irradiated at –196 °C. The analysis of the cell proliferative activity using Ki67 protein showed that cells irradiated at a dose of 50 Gy at a temperature of +22 °C completely lost their ability to proliferate. The proliferative activity of cells irradiated at the same dose, but at a temperature of –196 °C, was significantly reduced, but some of the cells (3.5 ± 1.1 %) still retained the ability to proliferate. After irradiation with a dose of 500 Gy at –196 °C, the cells completely lost their ability to proliferate, but partially retained the ability to adhere. The integral fluorescence of conjugated with the flurochrome γH2AX foci in MSCs irradiated at a dose of 500 Gy at a temperature of –196 °C was 1.8 times lower than that in MSCs irradiated at a temperature of +22 °C. Conclusion: The results of the study indicate that MSCs cryopreserved in a medium containing 10 % DMSO irradiated at liquid nitrogen temperature (–196 °C) can tolerate the effects of exposure to high doses (up to 50 Gy) of ionizing radiation. However, there is a rather high yield of residual DNA DSBs and a very low proliferative activity, which makes cells unsuitable for use in clinical practice. It seems promising to use a quantitative analysis of γH2AX foci to assess genome damage and the functional state of cells irradiated in a cryopreserved state.
mesenchymal stem cells, cryopreservation, DNA double-strand breaks, cell proliferation, bremstrahlung, ultrahigh doses
Введение
Изучение эффектов воздействия ионизирующего излучения (ИИ) в криоконсервированных клетках, облученных при температуре жидкого азота (–196 °С), представляет интерес для понимания механизмов действия ИИ. При столь низкой температуре резко снижаются процессы диффузии молекул, что приводит к существенному увеличению времени жизни свободных радикалов [1, 2]. Свободные радикалы, образующиеся при радиолизе воды, оказываются пространственно «заперты» и не могут взаимодействовать с находящимися на расстоянии биологическими макромолекулами. Это дает уникальные возможности для детальных исследований механизмов прямого (поглощение энергии биологическими молекулами-мишенями) действия ИИ. С другой стороны, современные технологии криоконсервации позволяют хранить соматические и половые клетки в течение десятилетий. Футурологи обсуждают возможность хранения криоконсервированных клеток в течение сотен лет и даже тысячелетий для полетов к другим звездным системам. При этом возникает вопросы: какие максимальные дозы ИИ выдерживают криоконсервированные клетки, и к каким эффектам приводит их облучение в больших дозах? Особый интерес для понимания механизмов повреждаемости криоконсервированных облученных клеток вызывают особенности образования критических радиационно-индуцированных повреждений ДНК – двунитевых разрывов (ДР). К сожалению, в открытой литературе отсутствуют данные как о количественном выходе ДР ДНК в клетках, облученных при температуре жидкого азота, так и об эффективности репарации этих повреждений после разморозки клеток.
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