Молекулярные механизмы регуляции продукции оксида азота в эпителиальных клетках мочевого пузыря лягушки
Оксид азота (NO) является важнейшим ауто/паракринным регулятором огромного спектра физиологических реакций. Низкомолекулярное и не несущее заряда соединение NO способно быстро диффундировать и свободно проникать через плотные клеточные слои и межклеточное пространство, не требуя специальных переносчиков. Внутриклеточные эффекты NO включают в себя его связывание с гем-содержащими белками… Читать ещё >
Содержание
- Список сокращений
- 1. ОБЗОР ЛИТЕРАТУРЫ
- 1. 1. Физиологическая роль оксида азота
- 1. 2. Биосинтез оксида азота
- 1. 3. Функциональная роль N0 в регуляции транспорта воды в собирательных трубках почки млекопитающих и мочевом пузыре амфибий
- 1. 4. Участие N0 в неспецифическом иммунном ответе в уроэпителии и почке млекопитающих
- 1. 5. Регуляция активности разных изоформ NOS
- 1. 5. 1. Регуляция конститутивных NOS
- 1. 5. 2. Механизмы регуляции iNOS
- 1. 5. 2. 1. Механизм действия ЛПС’на экспрессию iNOS
- 1. 5. 2. 2. Роль циклооксигеназы и простагландинов в индукции экспрессии iNOS
- 1. 5. 3. Регуляция продукции NO за счет изменения доступности субстрата
- 1. 5. 3. Л. Транспорт аргинина
- 1. 5. 3. 2. Аргиназа
- 2. 1. Животные
- 2. 2. Получение суспензии клеток мукозного эпителия мочевого пузыря лягушки
- 2. 3. Получение первичной культуры клеток мукозного эпителия мочевого пузыря лягушки
- 2. 4. Определение концентрации нитритов в культуральной среде
- 2. 5. Исследование активности NO-синтазы в гомогенате эпителиальных клеток мочевого пузыря лягушки
- 2. 6. Определение активности NOS и аргиназы в суспензии изолированных эпителиальных клеток
- 2. 7. Определение параметров транспортаZ-аргинина
- 2. 8. Измерение активности аргиназы в гомогенатах тканей и клеток
- 2. 9. Измерение концентрации белка в лизате эпителиальных клеток
- 2. 10. Выделение РНК из эпителиальных клеток мочевого пузыря лягушки
- 2. 11. Обратная транскрипция — полимеразная цепная реакция (ОТ-ПЦР)
- 2. 12. Электрофорез РНК и ДНК в агарозном геле
- 2. 13. Иммуноблоттинг
- 2. 13. 1. Приготовление проб для электрофоретического разделения
- 2. 13. 2. Электрофорез белков в полиакриламидном геле
- 2. 13. 3. Иммуноблоттинг со специфичными антителами
- 2. 14. Выделение лейкоцитов из крови лягушки и крысы
- 2. 15. Получение перитонеальных макрофагов крысы
- 2. 16. Статистическая обработка результатов
- 3. 1. NO-синтазы в эпителиальных клетках мочевого пузыря лягушки
- 3. 1. 1. Обнаружение NO-синтазной активности в изолированных клетках
- 3. 1. 2. Идентификация изоформ NOS
- 3. 1. 2. 1. Исследование кальциевой зависимости NOS в клеточных гомогенатах
- 3. 1. 2. 2. Влияние селективных ингибиторов конститутивных и индуцибельной NOS на NO-синтазную активность в эпителиальных клетках
- 3. 1. 2. 3. Идентификация изоформ NOS в эпителии мочевого пузыря лягушки методом иммуноблоттинга
- 3. 1. 2. 4. Идентификация iNOS методами ОТ-ПЦР и сиквенирования. Частичная характеристика первичной структуры iNOS
- 3. 2. 1. Влияние бактериального эндотоксина ЛПС Е. coli на продукцию нитритов изолированными эпителиальными клетками
- 3. 2. 2. 0. бнаружение рецептора TLR4 в эпителиальных клетках мочевого пузыря
- 3. 2. 3. Оценка вклада фагоцитирующих клеток в эффект ЛПС на продукцию нитритов
- 3. 2. 4. Изменение экспрессии мРНК и белка iNOS под действием ЛПС Е. col
- 3. 4. 1. Транспорт аргинина в клетках эпителия мочевого пузыря лягушки
- 3. 4. 1. 1. Характеристика транспортера катионных аминокислот в эпителиальных клетках мочевого пузыря лягушки
- 3. 4. 1. 2. Влияние ЛПС Е. coli на вход аргинина в клетки
- 3. 4. 2. Участие аргиназы в регуляции продукции NO
- 3. 4. 2. 1. Активность аргиназы в эпителиальных клетках и ее кинетические характеристики
- 3. 4. 2. 2. Идентификация изоформы аргиназы, присутствующей в эпителиальных клетках
- 3. 4. 2. 3. Активность NOS в условиях ингибирования аргиназы
- 3. 4. 2. 4. Конкурентные отношения NOS и аргиназы в присутствии ЛПС
- 3. 4. 2. 5. Влияние ЛПС на активность и экспрессию аргиназы
Список сокращений
АВТ — аргинин-вазотоцин
АДГ — антидиуретический гормон
БСА — бычий сывороточный альбумин
ДТТ — дитиотриитол
ЛПС — липополисахарид
НАДФН — никотинамидадениндинуклеотидфосфат
ПГЕ2 — простагландин Е nTF2a — простагландин F2a
ПГ12- простагландин
ТСХ — тонкослойная хроматография
ТХУ — трихлоруксусная кислота
ФАД — флавинадениндинуклеотид
ФМН — флавинаденинмононуклеотид
ФСБ — фосфатно-солевой буфер цАМФ — циклический аденозинмонофосфат цГМФ — циклический гуанозинмонофосфат
ЭДТА — этилендиаминтетрауксусная кислота
ВЕС — (8)-(2-бороноэтил)-Х-цистеин
ВН4 — тетрагидробиоптерин
СаМ — кальмодулин
CAT — транспортер катионных аминокислот (cationic amino acid transporter)
СОХ — циклооксигеназа (cyclooxygenase)
L-NAME — NG-nitro-Z,-arginine methyl ester
NFkB — нуклеарный фактор кВ (nuclear factor кВ)
PMSF — фенилметансульфонилфторид (phenylmethanesulfonylfluoride)
SDS — додецилсульфат натрия (sodium dodecyl sulfate)
TNFa — фактор некроза опухоли a (tumor necrosis factor a)
TLR — Toll-like receptor
NOS — NO-синтаза (nitric oxide synthase) eNOS, NOS3 — эндотелиальная NO-синтаза nNOS, NOS1 — нейрональная NO-синтаза iNOS, NOS2 — индуцибельная NO-синтаза Arg — аргинин
Asp — аспарагиновая кислота
Glu — глутаминовая кислота
Leu — лейцин
Lys — лизин
Met — метионин
Phe — фенилаланин
Ser — серин
Туг — тирозин
Val — валин
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