Medical Radiology and radiation safety Medical Radiology and radiation safety Медицинская радиология и радиационная безопасность 1024-6177 2618-9615 43694 10.12737/1024-6177-2020-65-5-12-20 Радиационная биология Radiation biology Радиационная биология Three-Color FISH Method: Dose-Effect Curves for Translocations in Peripheral Blood Lymphocyte Cultures after Gamma-Irradiation In Vitro ТРЁХЦВЕТНЫЙ FISH-МЕТОД: КРИВЫЕ ДОЗА-ЭФФЕКТ ДЛЯ ТРАНСЛОКАЦИЙ В КУЛЬТУРАХ ЛИМФОЦИТОВ ПЕРИФЕРИЧЕСКОЙ КРОВИ ПОСЛЕ ГАММА-ОБЛУЧЕНИЯ IN VITRO Нугис В. Ю. Nugis V. Yu.

доктор биологических наук;

doctor of sciences in biology;

 Снигирёва Г П Snigiryova G P Ломоносова Е. Е. Lomonosova E. E. Козлова М. Г. Kozlova M. G. Никитина В. А. Nikitina V. A.

кандидат медицинских наук;

candidate of medical sciences;

 Федеральный медицинский биофизический центр им. А.И. Бурназяна ФМБА России Москва Россия A.I. Burnasyan Federal Medical Biophysical Center of FMBA Moscow Russian Federation Федеральное государственное автономное учреждение «Национальный медицинский исследовательский центр нейрохирургии имени академика Н.Н. Бурденко» Министерства Здравоохранения Российской Федерации, Москва Россия Burdenko National Medical Research Center of Neurosurgery of the Ministry of Healthcare of the Russian Federation, Moscow Russian Federation Федеральный медицинский биофизический центр им. А.И. Бурназяна ФМБА России Москва Россия A.I. Burnasyan Federal Medical Biophysical Center of FMBA Moscow Russian Federation Федеральный медицинский биофизический центр им. А.И. Бурназяна ФМБА России Москва Россия A.I. Burnasyan Federal Medical Biophysical Center of FMBA Moscow Russian Federation Федеральный медицинский биофизический центр им. А.И.Бур­на­зяна ФМБА России Москва Россия A.I. Burnasyan Federal Medical Biophysical Center (FMBC) FMBA Moscow Russian Federation 65 5 12 20 https://naukaru.ru/en/nauka/article/43694/view

Цель: Построение кривых доза-эффект для транслокаций, выявленных с помощью трёхцветного FISH-метода, по результатам цитогенетического анализа культур лимфоцитов периферической крови здоровых доноров после гамма-облучения in vitro. Материал и методы: Использовалась венозная кровь трёх доноров (2 мужчин и 1 женщина в возрасте от 28 лет до 41 года), подвергнутая гамма-облучению in vitro от источника 60Со в дозах 0,10; 0,15; 0,25; 0,35; 0,50; 0,75; 1,00; 1,50; 2,00 и 3,00 Гр при температуре 37ºС (мощность дозы 0,5 Гр/мин). Для трёхцветного FISH-окрашивания использовались два разных набора ДНК-зондов к 1, 4, 12 и 2, 3, 8 парам хромосом. Для анализа отбирали метафазы с квазидиплоидным числом хромосом (40-46) и полным набором всех FISH-окрашенных хромосом с учётом всей их суммарной длины. Также производилась дифференциация стабильных и нестабильных клеток. При цитогенетическом анализе применялась традиционная терминология с обозначением транслокаций как реципрокных (полных, двухсторонних), нереципрокных (терминальных, неполных или односторонних) или интерстициальных. Результаты: Полученные численные данные были использованы для статистического сравнения частот FISH-регистрируемых транслокаций при использовании разных наборов ДНК зондов, при подсчёте аберраций хромосом во всех (нестабильных и стабильных) и стабильных метафазных клетках, сопоставлении частот FISH-регистрируемых транслокаций и дицентриков и оценке вклада уровня транслокаций между FISH-окрашенными парами хромосом в общую частоту транслокаций. Построенные кривые доза-эффект в целом соответствовали линейно-квадратичному виду. Заключение: Дозовые зависимости, полученные для транслокаций при использовании двух разных выбранных трёхцветных наборов ДНК-зондов статистически значимо не различались между собой. При этом цитогенетический анализ только стабильных метафазных клеток выявлял тенденцию к регистрации более низких уровней транслокаций, чем при анализе всех клеток (нестабильных и стабильных), при наиболее высоких дозах 2 и 3 Гр. Уровни дицентриков, образованных с участием FISH-окрашенных хромосом, были существенно ниже числа наблюдаемых транслокаций. Количественный вклад транслокаций между FISH-окрашенными парами хромосом оказался очень низким, что явно не способствует увеличению чувствительности FISH-метода ретроспективной оценки дозы по сравнению с его одноцветным вариантом. В то же время трёхцветная FISH-окраска позволяет выявлять такие варианты хромосомных перестроек, которые не регистрируются с помощью одноцветного FISH-метода.

Purpose: Plotting dose-effect curves for translocations identified using the tricolor FISH method based on the results of cytogenetic analysis of cultures of peripheral blood lymphocytes of healthy donors after in vitro gamma irradiation. Material and methods: Venous blood was obtained from three donors (2 men and 1 woman aged from 28 to 41 years) and subjected to in vitro gamma irradiation from a 60Co source at doses of 0.10; 0.15; 0.25; 0.35; 0.50; 0.75; 1.00; 1.50; 2.00 and 3.00 Gy at 37 ° C (dose rate 0.5 Gy / min). For tricolor FISH staining, two different sets of DNA probes were used for chromosome pairs 1, 4, 12 and 2, 3, 8. Metaphases with a quasi-diploid number of chromosomes (40-46) and a complete set of all FISH-stained chromosomes, taking into account their total length, were selected for analysis. Differentiation of stable and unstable cells was also carried out. In the cytogenetic analysis, traditional terminology was used with the designation of translocations as reciprocal (complete, two-sided), non-reciprocal (terminal, incomplete, or unilateral), or interstitial. Results: The obtained numerical data were used to statistically compare the frequencies of FISH-recorded translocations when using different sets of DNA probes, when calculating of chromosome aberrations were in all (unstable and stable) and stable metaphase cells, when comparing of the frequencies of FISH-recorded translocations and dicentrics, and assessing of the contribution of the level of translocations between FISH-stained chromosome pairs in the total translocation frequency. The plotted dose-effect curves generally corresponded to the linear-quadratic form. Conclusion: Dose dependences obtained for translocations using two different selected tricolor sets of DNA probes did not differ statistically significantly. At the same time, cytogenetic analysis of only stable metaphase cells revealed a tendency to register lower levels of translocations than when analyzing all cells (unstable and stable ), at the highest doses of 2 and 3 Gy. The levels of dicentrics formed with the participation of FISH-stained chromosomes were significantly lower than the number of observed translocations. The quantitative contribution of translocations between FISH-stained pairs of chromosomes turned out to be very low, which clearly does not contribute to an increase in the sensitivity of the FISH method of retrospective dose estimation as compared to its one-color version. At the same time, the three-color FISH-staining makes it possible to identify such variants of chromosomal rearrangements that are not recorded using the one-color FISH method.

 культура лимфоцитов периферической крови гамма-облучение in vitro трёхцветный FISH-метод транслокации дозовые зависимости peripheral blood lymphocytes culture gamma-irradiation in vitro three-color FISH method translocations dose-effect curves

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