Estudo dos mecanismos de ação do G-CSF e do AMD3100 na mobilização de células-tronco e produção de citocinas em cultura primária

Abstract

The bone marrow is the mean site described as reserve subpopulations of adult stem cells, including hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC). Different factors potentially stimulate mobilization of these stem cells into peripheral blood. The G-CSF and AMD3100 have been shown to act by disrupting CXCR4/SDF-1α retention axis, stimulating mobilization of HSC and MSC to the bloodstream. The aim of this study was to evaluate the influence of G-CSF and AMD3100 in HCS and MSC mobilization to the peripheral blood and in the cytokine profile produced by these cells in vitro. For this experiment, we were used 5 groups with 09 male mice, BALB-c. HSC and MSC were identified through specific cell markers by flow cytometry. The HSC and MSC samples, from peripheral blood (PB) and bone marrow (BM) were cultured in vitro and cytokines were measured in supernatants of these cultures by flow Cytometry CBA (Cytometric Bead Array System). Statistical analyzes were performed with the GraphPad Prism 5.0. In results we observed the statistically significant difference in HSC mobilization in mice treated with PBS that received one dose of AMD3100 and the animals treated with G-CSF over 04 successive days. Regarding the MSC, was observed a statistically significant difference in mobilization only in the group of animals treated with G-CSF. In the bone marrow cultures with or without G-CSF stimulus we observed adherent cells with fibroblastoid morphology. In the cultures of peripheral blood was observed that the cells had this same morphology when they were stimulated with G-CSF. There was an increased production of IL-6, TNF-α, IFN-γ and IL-10 in early cultivation of the SC stimulated with G-CSF, in the cultures of bone marrow cells and peripheral blood. Increased IL-2 and IL-4 cytokines was found in some groups in the late stage of the cultivation. IL-17A had different concentrations, maintaining a stable production in different groups and days of cultivation. Thus, it can be suggested that AMD3100 induces a rapid mobilization of CD34+ cells into the circulation and that G-CSF is directly or indirectly involved in the mobilization of stem cells from bone marrow to peripheral blood, as is kwown, but also in the differentiation of MSC, modulation process and production of several other cytokines.