Мультифотонная микроскопия и масс-спектрометрия в прижизненном выявлении метаболической гетерогенности гепатоцитов

Цель исследования — оценить возможность выявления гетерогенности гепатоцитов по метаболическому статусу в нормальной печени с применением современных методов мультифотонной микроскопии и масс-спектрометрии.

Материалы и методы. Степень гетерогенности гепатоцитов по общей метаболической активности оценивали с применением метода мультифотонной микроскопии на основе данных об интенсивности автофлуоресценции внутриклеточных кофакторов НАД(Ф)Н и ФАД. Гетерогенность гепатоцитов по интенсивности внутриклеточных метаболических процессов определяли с помощью метода время-разрешенного имиджинга (FLIM — fluorescence lifetime imaging) на основе данных о вкладах времен жизни флуо­ресценции различных форм кофактора НАД(Ф)Н. Методом времяпролетной масс-спектрометрии вторичных ионов (TоF-SIMS — time-of-flight secondary ion mass spectrometry) проведено исследование липидного и аминокислотного состава гепатоцитов.

Результаты. Методом мультифотонной микроскопии выявлена гетерогенность гепатоцитов по общей метаболической активности. С применением FLIM установлена высокая степень гетерогенности по интенсивности процессов окислительного фосфорилирования, гликолиза и синтетических процессов (липогенеза, синтеза нуклеиновых кислот и пентозофосфатного пути). Методом TоF-SIMS выявлено наличие гетерогенности гепатоцитов по аминокислотному и липидному составам, что указывает на различную интенсивность синтетических процессов отдельных гепатоцитов. Кроме того, установлены различия в содержании PO₃-ионов. Результаты TоF-SIMS-исследования согласуются с данными мультифотонной микроскопии и FLIM, подтверждая выявленную гетерогенность гепатоцитов по общей метаболической активности, а также по интенсивности внутриклеточных метаболических процессов.

Заключение. Используемые в работе новейшие методы флуоресцентного биоимиджинга и масс-спектрометрии позволяют эффективно выявлять гетерогенность гепатоцитов по метаболическому статусу. Наличие гетерогенности необходимо учитывать при анализе ткани печени в различных состояниях с применением методов флуоресцентного биоимиджинга.

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