Обоснование применения мезенхимальных стволовых клеток, полученных in vitro у детей с гематологическими и онкологическими заболеваниями тема диссертации и автореферата по ВАК РФ 14.01.21, кандидат медицинских наук Устюгов, Андрей Юрьевич

• Устюгов, Андрей Юрьевич
• кандидат медицинских наук
• 2010

Оглавление диссертации кандидат медицинских наук Устюгов, Андрей Юрьевич

ВВЕДЕНИЕ.

Глава 1. ОБЗОР ЛИТЕРАТУРЫ.

1.1 Мезенхимальные клетки и стромальное микроокружение.

1.2 Роль мезенхимальных клеток в гомопоэзе.

1.3 Иммунофенотипическая идентификация МСК.

1.4 Методы выделения МСК.

1.5 Свойства МСК.

1.6 Иммуномодулирующие свойства МСК.

1.7 Применения МСК in vivo:.

1.7.1 Применение МСК у животных

1.7.2 Применение МСК у людей

1.8 Безопасность применения.

1.9 Иммуногенетика.

1.10 Физиологическая роль МСК.

Глава 2. МАТЕРИАЛЫ И МЕТОДЫ.

2.1 Образцы КМ для культивирования МСК.

2.2Дети, получавшие МСК для ко-трансплантаций.

Дети, получавшие МСК для контроля РТПХ

2.3 Образцы КМ для оценки химеризма МСК.

2.4 Клоногенное культивирование стромальных фибробластов костного мозга

2.5 Индукция дифференцировки мезенхимальных предшественников.

2.6 Иммунофенотипирование методом проточной цитофлюорометрии.

2.7 Изучение динамики роста МСК при длительном культивировании.

2.8 Определение зависимости содержания стромальных клеток в культуре от времени доставки образца костного мозга в лабораторию.

2.9 Приготовление цитогенетических препаратов.

2.10 Кариотипирование.

2.11 Анализ частоты анеуплоидии.

2.12 Клоногенное культивирование стромальных фибробластов костного мозга для изучения химеризма.

2.13 Методика определения химеризма (метод типирования БТЯ-локусов).

2.14 Бактериологическое исследование МСК.

2.15 Определение ДНК цитомегаловируса человека (СМУ).

2.16 Статистическая обработка данных.

Глава 3. Результаты и их обсуждение.

ЗЛМорфологическое подтверждение используемой нами методики выделения и культивирования МСК.

3.2Дифференцировочные потенции клеток получаемой нами культуры МСК костного мозга.

З.ЗСветооптические характеристики МСК при длительном культивировании.

3.4 Влияние сроков культивирования на фенотипический состав монослойной культуры.

3.5Интенсивность экспрессии, характерных для МСК антигенов при культивировании.

З.бДинамика роста клеточной популяции.

3.7Определение зависимости содержания стромальных клеток в культуре от времени доставки костного мозга в лабораторию.

3.8Кариотипирование мононослойной культуры костного мозга.

3.9Частота анеуплоидии МСК.

3.10 Бактериологическое исследование МСК.

3.11 Определение ДНК цитомегаловируса человека (СМУ).

3.12 Клиническое применение культивируемых МСК у детей.

ЗЛЗИсследование химеризма МСК костного мозга у пациентов, перенесших аллогенную трансплантацию КМ.

3.14 Результаты линейного химеризма.

Выводы.

Заключение диссертации по теме «Гематология и переливание крови», Устюгов, Андрей Юрьевич

Выводы

1. Полученные при помощи усовершенствованной методики культивирования in vitro МСК КМ показали возможность приживления при трансплантациях у детей с гематологическими и онкологическими заболеваниями.

2. МСК, полученные в результате усовершенствованной методики культивирования in vitro, сохраняют полноценную функциональную активность, способность к дифференцировке в адипоциты и остеоциты.

3. Для стандартной оценки МСК в трансплантационном материале предложена следующая иммунофенотипическая панель: CD105, CD166, CD44, CD73, CD13, CD29, CD90.

4. При длительном культивировании in vitro МСК КМ человека 3-4 пассажа обладают высокой пролиферативной активностью, которая снижается при дальнейшем культивировании и становится минимальной к 10-12 пассажу.

5. После 3 пассажа в клеточной популяции отсутствовали клетки с гемопоэтическим и эндотелиальном фенотипом (CD45, CD34, CD 133, CD3, CD 19, CD25, CD38, CD45, CD106, CD31). Наблюдалась высокая экспрессия маркеров МСК KM (CD90, CD 105, CD 166, CD44, CD73, промежуточная CD 13 и СТ)29.).При увеличении срока культивирования МСК до 10-12 пассажа снижается количество клеток, экспрессирующих CD90, CD 105 и CD166. Наблюдается тенденция к снижению CD29, CD13, а также исчезают примеси гемопоэтических клеток (CD45+), эндотелиальных клеток (CD31+) и моноцитов (CD 14+)

6. В результате длительного культивирования у МСК КМ на ранних и поздних пассажах хромосомный набор не менялся (46,XY или 46,XX) и отсутствовали анеуплоидии.

7. Полученные нами данные оценки трансфузионной безопасности у детей позволяют предположить, об отсутствии ранних трансфузионных осложнений при применении приготовленных in vitro МСК у детей.

8. Используемый метод ПЦР определения химеризма МСК КМ продемонстрировал достаточную информативность и может применяться для оценки приживления МСК у детей с гематологическими и онкологическими заболеваниями.

Практические рекомендации

1. Метод может быть рекомендован для экспансии ex vivo МСК КМ в клинических целях

2. Для культивирования МСК in vitro рекомендованы образцы костного мозга, время у которых от момента забора костного мозга донора до начала выделения и культивирования прошло не более 6 часов.

3. Для оценки иммунофенотипа МСК в трансплантационном материале рекомендуется использовать панель, состоящую из характерных для МСК маркеров (CD90, CD105, CD166, CD44, CD73, CD13, CD29), так и маркеров, специфичных для гемопоэтических клеток (CD3, CD14, CD 19, CD25, CD29, CD31, CD34, CD38, CD45, CD 106, HLA-DR).

4. Для клинических целей рекомендовано применять МСК 3-4 пассажа.

5. В случае необходимости использования в терапевтических целях МСК более поздних сроков культивирования необходим строгий контроль поверхностного фенотипа и генетической стабильности клеточных трансплантатов.

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