63ª Reunião Anual da SBPC

C. Ciências Biológicas - 3. Bioquímica - 5. Química de Macromoléculas

A PROTEOMIC STUDY ON OSTEOBLASTIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS BY iTRAQ LABELING

Leonardo Barcelos de Paula 1 Fabíola Singaretti de Oliveira 2 Adalberto Luiz Rosa 2 José César Rosa 3

1. Depto. de Biologia Celular, Molecular e Bioagentes Patogênicos, Fac. de Medicina de Ribeirão Preto – FMRP-USP 2. Laboratório de Cultura de Células, Fac. de Odontologia de Ribeirão Preto – FORP-USP 3. Prof. Dr./Orientador - Depto. de Biologia Celular, Molecular e Bioagentes Patogênicos – FMRP-USP

INTRODUÇÃO:

The growth, development and maintenance of bone tissue are highly regulated processes. Several proteins such as hormones, growth factors and cytokines are actively involved in these processes and exert direct activity on osteoblastic and osteoclastic cells, acting in their differentiation and metabolic activation. The process of bone regeneration is initiated by local stimulating factors as bone morphogenetic proteins (BMP). BMPs are a product of the metabolism of osteoblasts, odontoblasts and various tumor cells and is stored in the form of concentrates in bone, dentin and neoplastic cells of osteosarcoma and certain odontogenic tumors such as fibroma cementifying, cementoblastoma benign dentinoma, odontogenic fibroma and odontoma. Clarify the mechanisms that control bone remodeling is a very relevant issue. Accordingly, the mesenchymal stem cells have attracted great interest because of its potential involvement in the process of tissue repair. Obtaining functional osteoblasts from mesenchymal stem cells has been used in tissue engineering and cell therapy.

METODOLOGIA:

Thus, this present work performed a proteomic analysis of proteins involved in various stages of osteoblast differentiation of mesenchymal stem cells from bone marrow of Wistar rat and human, in order to obtain more information on the biology of cell differentiation and bone tissue. Mesenchymal stem cells obtained from bone marrow were cultured in osteogenic medium for different periods to obtain cells at different stages of osteoblast differentiation. For proteomics analysis tools were used as the strategy of shotgun proteomics and relative quantification (iTRAQ - isobaric Tag for Relative and Absolute quantitation) for protein separation and mass spectrometry to identify proteins.

RESULTADOS:

In this context, our results take us to conclude that the MSCs of Wistar rat bone marrow express genes that are involved in osteogenic differentiation in vitro when stimulated to form bone matrix during the 14 days, ie stimulating factor in the microenvironment is of fundamental importance, the MSCs from human bone marrow showed similar results with rat MSCs at the genomic level during osteogenic differentiation, however, when stimulated in vitro formed bone matrix within 21 days, using two proteomic approaches, we could identify proteins important that are involved in the process of differentiation. But it should be noted that although it has been identified genes that seem involved in the process of differentiation, it was not reflected in the proteome of these cells at 7 and 14 days after induction of the osteogenic differentiation, indicating that most of the functionality of these cells and other biological processes are preserved, such as cell proliferation remained without major changes. This indicates that manipulations of isolation, culture and induction of differentiation of these cells did not affect the proteome, with positive aspects to the use of MSCs in cell therapy.

CONCLUSÃO:

From the methodological point of view, this work opens up the use of proteomic strategies based on the score for isobars in combination with protein separation by electrophoresis, one-dimensional SDS-PAGE for the analysis of complex biological samples and limited quantities of production as MSCs. The study of protein expression during stages of osteoblast differentiation of MSCs from bone marrow should reflect their functional status and contribute to the understanding of pathways involved in the process of differentiation.

Palavras-chave: Mesenchymal stem cells, Osteoblast differentiation, Shotgun proteomics and iTRAQ.