Анализ молекулярных аспектов функционирования миозина II в мышечном сокращении и клеточной подвижности с помощью белка KRP
Диссертация
В норме клеточная миграция происходит на всем протяжении эмбриогенеза, в постнатальном развитии, репарации и ремоделировании тканей, при ангиогенезе, воспалительном и иммунном ответах и других биологических процессах. Нарушения миграционного поведения клеток часто сопровождают различные патологические состояния, в том числе в сердечно-сосудистой системе, при заживлении ран и канцерогенезе… Читать ещё >
Содержание
- СПИСОК СОКРАЩЕНИЙ
- СПИСОК ИЛЛЮСТРАЦИЙ
- ОБЗОР ЛИТЕРАТУРЫ
- 1. ОБЩИЕ ПРИНЦИПЫ АКТОМИОЗИН-ЗАВИСИМОЙ КЛЕТОЧНОЙ ПОДВИЖНОСТИ И СОКРАТИМОСТИ ГЛАДКИХ МЫШЦ
- 1. 1. Актомиозиновый цитоскелет — механическая основа движения клеток
- 1. 2. Актин и миозин II — главные сократительные белки клетки
- 1. 3. Фосфорилирование РЛЦ — ведущий механизм активации миозина
- 1. 4. Ферменты, регулирующие уровень фосфорилирования миозина II
- 1. 4. 1. Киназа легких цепей миозина (КЛЦМ)
- 1. 4. 2. Rho-активируемая киназа (Rho-киназа)
- 1. 4. 3. Другие Са"' -независимые протеинкиназы
- 1. 4. 4. Фосфатаза легких цепей миозина (ФЛЦМ)
- 1. 4. 5. Химические ингибиторы ферментов-модуляторов активности миозина II
- 2. 1. Ультраструктура миозиновых филаментов II гладких мышц
- 2. 2. Факторы, препятствующие деполимеризации миозиновых филаментов гладких мышц
- 2. 3. Особенности регуляции сокращения гладких мышц под действием фосфорилирования РЛЦ миозина
- 2. 4. Пермеабилизованные гладкомышечные волокна как модель исследования регуляции сокращения гладкой мускулатуры
- 3. 1. Двигательные реакции в миграции немышечных клеток
- 3. 2. Распределение структурных элементов миозина II в мигрирующей клетке
- 3. 3. Пространственная регуляция фосфорилирования РЛЦ миозина II в клетке
- 3. 4. Экспериментальные подходы к исследованию механизмов миозин II -зависимой подвижности немышечных клеток
- 4. 1. Структура KRP
- 4. 2. KRP как эндогенный регулятор активности миозина II
- 1. 1. Реактивы и материалы для биохимических и молекулярно-биологических исследований
- 1. 2. Реактивы и материалы для клеточно-биологических исследований
- 1. 3. Реактивы и материалы для физиологических исследований
- 2. 1. Биохимические методы
- 2. 1. 1. Определение концентрации белка
- 2. 1. 2. Электрофорез в полиакриламидном геле в присутствии додецилсульфата натрия (ДСН-электрофорез)
- 2. 1. 3. Мочевинно-глицеролъный электрофорез
- 2. 1. 4. Иммуноблоттинг
- 2. 1. 5. Приготовление образцов клеток и гладкомышечных волокон для ДСП-электрофореза
- 2. 1. 6. Приготовление образцов из гладкомышечных волокон и NIH3T3 фибробластов для мочевинно-глицеролъного электрофореза
- 2. 1. 7. Экспрессия и очистка рекомбинантного KRP и его мутантных форм
- 2. 1. 8. Фосфорилирование KRP in vitro
- 2. 1. 9. Получение KRP, меченого 5-TMRIA
- 2. 2. Молекулярно-биологические методы
- 2. 2. 1. Создание и характеристика молекулярно-генетических конструкций
- 2. 3. Клеточно-биологические методы
- 2. 3. 1. Культивирование фибробластов линии NIH ЗТЗ
- 2. 3. 2. Транзиторная трансфекция фибробластов линии NIH3T
- 2. 3. 3. Получение трансгенных фибробластов линии NIH ЗТЗ, стабильно экспрессирующих KRP и ACKRP
- 2. 3. 4. Направленная миграция в камере Бойдена
- 2. 3. 5. Адгезия фибробластов на иммунологические планшеты, покрытые коллагеном
- 2. 3. 6. Измерение миграции клеток методом зарастания царапины (Scratch assay)
- 2. 3. 7. Измерение сократительной активности фибробластов в коллагеновом матриксе
- 2. 3. 8. Экстракция растворимого миозина из клеток с помощью Тритона Х
- 2. 3. 9. Вычисление линейных параметров ядер и объема фибробластов линии ШНЗТЗ
- 2. 4. Физиологические методы
- 2. 4. 1. Получение препарата taenia caeci, пермеабшизованного Тритоном Х
- 2. 4. 2. Измерение сократительной активности taenia caec
- 2. 4. 3. Приготовление препаратов taenia caeci с меченым KRP для флуоресг (ентной микроскопии
- 2. 5. Количественная обработка изображений и статистический анализ
- 1. ВЛИЯНИЕ KRP НА СОКРАТИМОСТЬ СКИНИРОВАННЫХ ГЛАДКОМЫШЕЧНЫХ ВОЛОКОН
- 1. 1. Характеристика скинированных препаратов гладких мыщц taenia caec
- 1. 2. Влияние KRP на Са2±независимое сокращение, вызванное микроцистином
- 1. 3. Влияние KRP на Са2±зависимое сокращение и расслабление
- 1. 3. 1. Эффекты полноразмерного и фосфорилированного KRP на расслабление скинированных волокон
- 1. 3. 2. Релаксирующие эффекты мутантных форм KRP
- 2. 1. Получение и характеристика трансгенных по KRP фибробластов
- 2. 2. Влияние KRP на фосфорилирование РЛЦ миозина в фибробластах
- 2. 3. Влияние KRP на количество полимерного миозина II в фибробластах
- 2. 4. Роль С-концевого домена KRP в регуляции функционального состояния миозина II в клетках
- 2. 5. Влияние KRP на подвижность и сократимость трансгенных фибробластов
- 2. 6. С-концевая последовательность KRP необходима для стимуляции хемотаксиса фибробластов
- 2. 7. Внутриклеточные эффекты KRP связаны с миозином, регулируемым КЛЦМ и, возможно, Zip-киназой, но не Ию-киназой
- 2. 7. 1. Влияние KRP на фосфорилирование миозина при ингибировании киназ РЛЦ миозина
- 2. 1. 2. Влияние KRP на двигательные реакции клеток при ингибировании киназ РЛЦ миозина
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- Мне бы хотелось поблагодарить всех преподавателей кафедры Биохимии биологического факультета МГУ и заведующего кафедрой профессора Николая Борисовича Гусева за полученное мною образование.
- И, в заключении, мне бы хотелось выразить благодарность своей семье моим родителям и мужу за понимание, терпение и веру в мои силы.