Caracterização do meio de cultura de células tronco mesenquimais adiposas de cães
Data
2019-08-28
Autores
Silvestre, Priscila Modesto
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Introdução: O secretoma é definido como proteínas liberadas por uma célula, tecido ou organismo por meio de mecanismos de secreções clássicas e não clássicas. Até o momento, as proteínas secretadas com caracterização melhor, são proteínas liberadas pelas células tronco mesenquimais (CTMs), adiposas (CTMAs), neurais e embrionárias entre outras. As CTMAs podem ser facilmente isoladas em grandes quantidades a partir de tecido adiposo obtido por procedimentos cirúrgicos simples. Estas células expressam uma série de proteínas específicas e marcadores fenotipicamente semelhantes às CTMs. Assim como as CTMs, têm o potencial de se diferenciar em linhagens osteogênicas, condrogênicas, miogênicas, neuronais, cardiomiócitas, epiteliais e endoteliais. Avanços recentes no perfil molecular permitiram mapeamento de proteínas secretadas por vários tipos de células. As técnicas oferecem uma ligeira vantagem na detecção de proteínas menos abundantes em meios condicionados. Métodos baseados nas medições de intensidades de pico espectrais de massa foram introduzidos recentemente em conjunto com melhorias na tecnologia de espectrometria de massa e fornecem uma maneira conveniente e confiável em estudos proteômicos. Objetivo: O objetivo foi analisar o meio condicionado de CTMAs de cães, por meio da análise proteômica para identificar alvos expressos na solução e avaliar as suas funções. Métodos: As CTMAs foram isoladas a partir de tecido adiposo de cães, diferenciadas e caracterizadas por citometria de fluxo em terceira passagem com os marcadores CD34, CD45, CD90 e CD71. As análises labell free de espectrometria de massas foram realizadas por meio de um equipamento de nanocromatografia líquida Ultimate 3000 LC acoplado à um equipamento de espectrometria de massas Q-Exactive. A identificação das proteínas foram submetidas ao software PatternLab (versão 4.0.0.84). A análise da expressão no secretoma destas células permitiu avaliar relações com proliferação e interação celular, sinalizações, além de modulação do sistema imune e angiogênese. Este conhecimento possibilitará aplicações do secretoma e não mais das células tronco, minimizando assim possíveis riscos de imunogenicidade, questões éticas e regulatórias.
Introduction: Secretoma is defined as proteins released by a cell, tissue or organism through mechanisms of classical and non-classical secretions. So far, the best characterized secreted proteins are proteins released by mesenchymal stem cells (MSCs), adipose (MSCs), neural and embryonic stem cells among others. CTMAs can be easily isolated in large quantities from adipose tissue obtained by simple surgical procedures. These cells express a series of specific proteins and markers phenotypically similar to MSCs. Like MSCs, they have the potential to differentiate into osteogenic, chondrogenic, myogenic, neuronal, cardiomyocyte, epithelial and endothelial strains. Recent advances in molecular profiling have allowed mapping of proteins secreted by various cell types. The techniques offer a slight advantage in detecting less abundant proteins in conditioned media. Methods based on measurements of mass spectral peak intensities have recently been introduced in conjunction with improvements in mass spectrometry technology and provide a convenient and reliable way for proteomic studies. Objective: The objective was to analyze the conditioned medium of dog CTMAs by shotgun proteomic analysis to identify targets expressed in the solution and to evaluate their functions. Methods: CTMAs were isolated from dog adipose tissue, differentiated and characterized by third pass flow cytometry with markers CD34, CD45, CD90 and CD71. Labell free mass spectrometry analyzes were performed using an Ultimate 3000 LC liquid nanochromatography equipment coupled with a Q-Exactive mass spectrometry equipment. Protein identification was submitted to PatternLab software (version 4.0.0.84). The analysis of the expression in the secretome of these cells allowed to evaluate relations with cell proliferation and interaction, signaling, besides modulation of the immune system and angiogenesis. This knowledge will enable applications of the secretoma rather than stem cells, thus minimizing possible risks of immunogenicity, ethical and regulatory issues.
Introduction: Secretoma is defined as proteins released by a cell, tissue or organism through mechanisms of classical and non-classical secretions. So far, the best characterized secreted proteins are proteins released by mesenchymal stem cells (MSCs), adipose (MSCs), neural and embryonic stem cells among others. CTMAs can be easily isolated in large quantities from adipose tissue obtained by simple surgical procedures. These cells express a series of specific proteins and markers phenotypically similar to MSCs. Like MSCs, they have the potential to differentiate into osteogenic, chondrogenic, myogenic, neuronal, cardiomyocyte, epithelial and endothelial strains. Recent advances in molecular profiling have allowed mapping of proteins secreted by various cell types. The techniques offer a slight advantage in detecting less abundant proteins in conditioned media. Methods based on measurements of mass spectral peak intensities have recently been introduced in conjunction with improvements in mass spectrometry technology and provide a convenient and reliable way for proteomic studies. Objective: The objective was to analyze the conditioned medium of dog CTMAs by shotgun proteomic analysis to identify targets expressed in the solution and to evaluate their functions. Methods: CTMAs were isolated from dog adipose tissue, differentiated and characterized by third pass flow cytometry with markers CD34, CD45, CD90 and CD71. Labell free mass spectrometry analyzes were performed using an Ultimate 3000 LC liquid nanochromatography equipment coupled with a Q-Exactive mass spectrometry equipment. Protein identification was submitted to PatternLab software (version 4.0.0.84). The analysis of the expression in the secretome of these cells allowed to evaluate relations with cell proliferation and interaction, signaling, besides modulation of the immune system and angiogenesis. This knowledge will enable applications of the secretoma rather than stem cells, thus minimizing possible risks of immunogenicity, ethical and regulatory issues.
Descrição
Palavras-chave
Secretoma celular, Células tronco adiposas, Proteômica, Cellular secretoma, Adipose stem cells, Proteomics