Регуляция дифференцировки клеток трансгенными нейротрофическими факторами
Диссертация
Установлено, что нейроэктодерма трансфицированных эмбрионов дрозофилы способна стимулировать дифференцировку нервных клеток млекопитающих при их совместном культивировании на искусственных средах. При этом происходит направленное образование отростков у эмбриональных спинальных ганглиев, что является необходимым условием при трансплантации. Показано, что промоторно-регуляторная область гена ЪэрЮ… Читать ещё >
Содержание
- 1. ОБЗОР ЛИТЕРАТУРЫ
- 1. 1. Межвидовая ксенотрансплантация
- 1. 2. Лечение нейродегенеративных заболеваний методом трансплантации
- 1. 3. Восстановительный потенциал центральной нервной системы и связанные с ним проблемы трансплантологии
- 1. 3. 1. Формирование глиального рубца и его влияние на регенерацию аксонов
- 1. 3. 2. Характеристика методов, направленных на улучшение восстановительных свойств ЦНС при повреждениях
- 1. 4. Нейротрофические факторы (НФ)
- 1. 4. 1. Понятие и классификация нейротрофических факторов
- 1. 4. 2. Общая характеристика лигандов семейства ООМ^ОРЬб)
- 1. 4. 3. Характеристика рецепторов лигандов семейства ОБМ
- 1. 4. 4. Индуцируемая ОБМ7 ИЕТ-зависимая передача сигнала внутрь клетки
- 1. 4. 5. Индуцируемая ООМ7 ИЕТ-независимая передача сигнала внутрь клетки
- 1. 4. 6. Взаимодействие сигнального пути, индуцируемого вОМ7, с сигнальными путями других факторов
- 1. 4. 7. Экспрессия и физиологические функции ОБМ
- 1. 4. 8. Защитные и восстановительные свойства ОБМР
- 1. 4. 9. Общая характеристика лигандов семейства N0?
- 1. 4. 10. Характеристика фактора роста нервов
- 1. 4. 11. Структура фактора роста нервов
- 1. 4. 12. Рецепторы ИОЕ .V.-.>
- 1. 4. 13. Сигнальные каскады, запускаемые N0?
- 1. 4. 14. ВВ№- как член семейства N0?
- 1. 4. 15. Внутриклеточные каскады, активируемые BDNF
- 1. 4. 16. Исследование мышей, нокаутированных по гену BDNF
- 1. 4. 17. Свойства BDNF, влияние на дифференцировку нейронов
- 1. 4. 18. Влияние нейротрофических факторов на поведение и память
- 1. 4. 19. Нейротрофические факторы при нейродегенеративных заболеваниях.69 1.5. Белки теплового шока (Hsp)
- 1. 5. 1. Классификация Hsp
- 1. 5. 2. Факторы индукции Hsp
- 1. 5. 3. Регуляция экспрессии белков семейств Hsp
- 1. 5. 4. Локализация и свойства Hsp
- 1. 5. 5. Hsp70.v
- 1. 5. 6. Структурно-функционалный анализ белков семейства Hsp
- 1. 5. 7. Hsp и апоптоз
- 1. 5. 8. Стресс, адаптация и белки теплового шока
- 1. 5. 9. Перспективы использования системы Hsp в медицине
- 1. 5. 10. Экспрессия шаперонов в головном мозге
- 2. 1. Методы
- 2. 1. 1. Рестрикция плазмидной ДНК
- 2. 1. 2. Электрофорез в агарозном геле
- 2. 1. 3. Выделение фрагментов ДНК из геля
- 2. 1. 4. Лигирование
- 2. 1. 5. Приготовление компетентных клеток
- 2. 1. 6. Трансформация компетентных клеток плазмидами
- 2. 1. 7. Выделение плазмидной ДНК из Е. coli в малом объеме
- 2. 1. 8. Выделение плазмидной ДНК из E. coli в большом объеме
- 2. 1. 9. Контроль наличия вставки (блот-гибридизация по Саузерну)
- 2. 1. 10. Анализ ДНК-последовательности.Ill
- 2. 1. 11. Конструкции
- 2. 1. 12. Метод инъекций
- 2. 1. 13. Выведение линий
- 2. 1. 14. Гибридизация in situ на целых эмбрионах
- 2. 1. 15. Очистка плазмид для введения в культуру клеток Млекопитающих
- 2. 1. 16. Введение плазмиды в клетки линии НЕК293 при помощи ExGen 500 Transfection Reagent
- 2. 1. 17. Введение плазмиды в ЭСК мыши (линия R1) методом электропорации
- 2. 1. 18. Выделение суммарного препарата РНК
- 2. 1. 19. Проведение электрофореза РНК
- 2. 1. 20. Нозерн-блот гибридизация
- 2. 1. 21. Электрофорез в ПААГ
- 2. 1. 22. Вестерн-блот гибридизация
- 2. 1. 23. Культивирование клеток линии НЕК
- 2. 1. 24. Получение мышиных эмбриональных фибробластов
- 2. 1. 25. Желатинизирование ¡-культуральныхчашек
- 2. 1. 26. Обработка МЭФ митомицином С
- 2. 1. 27. Культивирование ЭСК мыши линии R
- 2. 1. 28. Проведение трансплантации
- 2. 1. 29. Получение срезов мозга
- 2. 1. 30. Иммуноцитохимический анализ
- 2. 1. 31. Количественная оценка величины глиального рубца
- 2. 1. 32. Ко-культивация фрагмента зачатка спинного мозга эмбриона человека с клетками трансгенной линии НЕК293, экспрессирующей gdnf
- 2. 1. 33. Получение первичной^культурьг клеток*D.melanogaster, трансгенных по гену ngf
- 2. 2. 1. Реактивы
- 2. 2. 2. Растворы, среды
- 2. 2. 3. Вектора
- 2. 2. 4. Праймера
- 2. 2. 5. Антитела
- 2. 2. 6. Линии клеток
- 3. 1. Создание трансгенных линий Drosophila melanogaster гомозиготных по нейральному гену млекопитающих
- 3. 2. Влияние экспрессии нейротрофического фактора на трансгенные клетки
- 3. 3. Влияние экспрессии нейротрофического фактора на окружающие клетки при ¡-кокультивации
- 3. 4. Влияние нейроэктодермы трансгенных линий на окружающие клетки при ксенотрансплантации
- 3. 5. Влияние белка теплового шока HSP70 дрозофилы на образование рубца при трансплантациях
- 3. 6. Использование промоторно-регуляторной (п/р) области гена белка теплового шока дрозофилы (Hsp70), в качестве регуляторного элемента при введении чужеродных генов в клетки млекопитающих
- 3. 7. Использование полученного вектора LP1, содержащего п/р область гена hsp70 дрозофилы <и маркерный ген gfp, в культурах клеток и в целых организмах
- 3. 8. Получение конструкции для экспрессии ngf в клетках млекопитающих
- 3. 9. Изучение влияния гиперэкспрессии ngf при ко-культивации
- 3. 10. Получение конструкции для экспрессии gdnf в клетках млекопитающих
- 3. 11. Исследование влияния гиперпродукции белка GDNF на трансгенные клетки
- 3. 12. Изучение влияния гиперэкспрессии ^о^-/при ко-культивации
- 3. 13. Изучение влияния гиперэкспрессии gdnf на образования глиальной ткани при трансплантациях
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