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Релокализация генов-партнеров по незаконной рекомбинации в условиях ингибирования ДНК-топоизомеразы II

Диссертация: Релокализация генов-партнеров по незаконной рекомбинации в условиях ингибирования ДНК-топоизомеразы II
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Лейкемии составляют 3% от общего числа всех онкологических заболеваний в мире. Четырьмя основными типами лейкемий являются острая лимфоцитарная лейкемия (ALL), хроническая лимфоцитарная лейкемия (CLL), острая мпелоидная ле11кемия (AML) п хроническая миелоидная лейкемия (CML). Развитие острой мпелоидной лейкемии обычно связывают с хролюсомнымн перестройками, приводящими к возникновению химерных… Читать ещё >

Содержание

  • список сокращений. введение. i. обзор литературы
    • 1. 1. Хромосомные транслокации, ассоциированные с онкологическими заболеваниями
      • 1. 1. 1. Незаконная рекомбинация
      • 1. 1. 2. История изучения механизмов незаконной рекомбинации
      • 1. 1. 3. Хромосомные транслокации и ассоциированные с ними заболевания
      • 1. 1. 4. Семейства химерных белков
      • 1. 1. 5. Гены-партнеры по хромосомным транслокациям и кластеризация точек разрыва ДНК
      • 1. 1. 6. Механизм возникновения хромосомных транслокаций
      • 1. 1. 7. Структура кластеров точек разрыва ДНК как предпосылка для образования хромосомных транслокаций
    • 1. 2. двухцепочечные разрывы ДНК и их репарация
      • 1. 2. 1. Репарация двухцепочечных разрывов ДНК
      • 1. 2. 1. ДНК-топоизомераза II и ее функции в клетке
      • 1. 2. 2. Ингибирование ДНК-топоизомеразы II как причина образования двухцепочечных разрывов ДНК
      • 1. 2. 3. Ингибирование ДНК-топоизомеразы II как предпосылка для возникновения хромосомных транслокаций
      • 1. 2. 4. Гипотезы возникновения хромосомных транслокаций
    • 1. 3. позиционирование хромосом в ядре и вероятность возникновения хромосомных транслокаций
      • 1. 3. 1. Хромосомные территории
      • 1. 3. 2. Пространственная близость хромосомных территорий способствует образованию транслокаций
      • 1. 3. 3. Перемешивание хромосомных территорий
      • 1. 3. 4. Современная модель организации хромосомных территорий
      • 1. 3. 5. Релокализация геномных локусов внутри ядра. Роль ядерных моторных белков в этих процессах. постановка задачи и методические подходы. ii. материалы и методы
    • II. 1. Материалы
  • II. 1.1. Клеточные линии
  • II. 1.2. Антитела
  • II. 1.3. Химические реактивы
  • II. 1.4. Программное обеспечение
    • II. 2. Методы
      • 11. 2. 1. Культивирование клеточных линий
      • 11. 2. 2. Электрофорез в пульсирующем поле
      • 11. 2. 3. Определение количества мертвых клеток
      • 11. 2. 4. Иммуноокрашивание
      • 11. 2. 5. Флюоресцентная гибридизация in situ (FISH)
        • 11. 2. 5. 1. Приготовление двумерных препаратов фиксированных клеток
        • 11. 2. 5. 2. Выделение бакмидной ДНК из клеток E. col
        • 11. 2. 5. 3. Синтез пробы
        • 11. 2. 5. 4. Гибридизация
        • 11. 2. 5. 5. Приготовление трехмерных препаратов клеток Jurkat
        • 11. 2. 5. 6. Гибридизация с пробой Vysis
      • 11. 2. 6. Компьютерная обработка изображений
      • 11. 2. 7. Статистический анализ данных
      • 11. 2. 8. Иммунопреципитация хроматина
        • 11. 2. 8. 1. Растворы
        • 11. 2. 8. 2. Методика
        • 11. 2. 8. 3. ПЦР в реальном времени с ТадМап-пробами. iii. результаты исследований
    • III. 1. Анализ взаимного расположения генов АМН и ЕТО в культуре первичных эмбриональных фибробластов человека
    • 111. 2. ингибирование лигирующей активности ДНК-топоизомеразы И вызывает перемещение гена ЕТО в направлении центра ядра
    • 111. 3. ингибирование ДНК-топоизомеразы ii вызывает релокализацию гена ЕТО в область ядрышка
    • 111. 4. ингибирование днк-топоизомеразы ii приводит к предпочтительному связыванию нуклеолина с bcr2 гена ЕТО. обсуждение результатов
  • выводы

Релокализация генов-партнеров по незаконной рекомбинации в условиях ингибирования ДНК-топоизомеразы II (реферат, курсовая, диплом, контрольная)

Лейкемии составляют 3% от общего числа всех онкологических заболеваний в мире. Четырьмя основными типами лейкемий являются острая лимфоцитарная лейкемия (ALL), хроническая лимфоцитарная лейкемия (CLL), острая мпелоидная ле11кемия (AML) п хроническая миелоидная лейкемия (CML). Развитие острой мпелоидной лейкемии обычно связывают с хролюсомнымн перестройками, приводящими к возникновению химерных генов. Транслокация t (8−21)(q22-q22), объединяющая гены АМЫ и ЕТО, известна как одна из наиболее частых хромосомных транслокаций, наблюдаемых при острой мпелоидной лейкемии. Ежегодно она диагносцируется в 40% случаев AML типа М2 (острая миелобластная лейкемия), а также сопровождает до 40% случаев детской AML, и вероятность ее возникновения практически не зависит от пола ребенка (Huret, 1997). Но особенно часто эта транслокация наблюдается при так называелнлх «обусловленных лечением» (treatment related) или вторичных лейкозах (t-AML).Исследования последних лет свидетельствуют о том, что во многих случаях к развитию вторичных лейкозов приводит противораковая химиотерапия «первичных» онкологических заболеваний, проводящаяся с использованием препаратов, специфически ингибирующих фермент ДНК-топоизомеразу П. ДНК-топоизомераза II является жизненно необходимым ферментом, так как катализирует топологические изменения в ДНК в ходе множества клеточных процессов, таких как сегрегация дочерних хромосом после завершения процесса репликации ДНК, транскрипция, рекомбинация и реорганизация хроматина. Именно поэтому при терапии онкологических заболеваний применяются препараты, ингибирующие ее активность и вызывающие гибель активно делящихся клеток. Вследствие сказанного представляется важным изучение механизмов возникновения хромосомных перестроек, возникающих вследствие ингибирования ДНК-топоизомеразы П. Знание этих механизмов должно не только внести существенный вклад в представления об организации и ф}Ч1кционпровании клеточного ядра, но и показать исследователям новые пути решения проблемы вторичных лейкозов.

1. Установлено, что в подавляющей части клеток, присутствующих в популяции первичных эмбриональных фибробластов человека (HEF 1698), гены AML1 и ЕТО сближены, но находятся в разных ядерных слоях.2. Продемонстрировано, что обработка клеток ингибиторами ДНК-топоизомеразы II приводит к перемещению внутри ядра гена ЕТО, в результате чего этот ген оказывается в одном ядерном слое с геном AML1, являющимся партнером гена ЕТО по хромосомным перестройкам.3. Показано, что обработка первичных эмбриональных фибробластов человека (HEF 1698) и клеток Jurkat этопозидом приводит к увеличению частоты ко локализации гена ЕТО с ядрышком.4. Продемонстрировано, что ингибирование ДНК-топоизомеразы II этопозидом в клетках Jurkat приводит к предпочтительному связыванию нуклеолина с Ьсг2 гена.

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