RESUMO
BACKGROUND: Macrophages are a functionally heterogeneous cell population and depending on microenvironments they polarize in two main groups: M1 and M2. Glutamic acid and glutamate receptors may participate in the regulation of macrophage plasticity. To investigate the role of glutamatergic systems in macrophages physiology, we performed the transfection of mGluR5 cDNAs into RAW-264.7 cells. RESULTS: Comparative analysis of modified (RAW-mGluR5 macrophages) and non-modified macrophages (RAW-macrophages) has shown that the RAW-mGluR5 macrophages absorbed more glutamate than control cells and the amount of intracellular glutamate correlated with the expression of excitatory amino acid transporters -2 (EAAT-2). Besides, our results have shown that RAW-mGluR5 macrophages expressed a higher level of peroxisome proliferator-activated receptor γ (PPAR-γ) and secreted more IL-10, high mobility group box 1 proteins (HMGB1) and Galectin-3 than control RAW-macrophages. CONCLUSIONS: We propose that elevation of intracellular glutamate and expression of mGluR5 may initiate the metabolic rearrangement in macrophages that could contribute to the formation of an immunosuppressive phenotype.
Assuntos
Plasticidade Celular/fisiologia , Macrófagos/fisiologia , Receptor de Glutamato Metabotrópico 5/fisiologia , Animais , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Galectina 3/análise , Galectina 3/metabolismo , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Proteína HMGB1/análise , Proteína HMGB1/metabolismo , Interleucina-10/análise , Interleucina-10/metabolismo , Lipopolissacarídeos , Camundongos , Óxido Nítrico/metabolismo , PPAR alfa/análise , PPAR alfa/metabolismo , Fenótipo , Células RAW 264.7 , Transfecção/métodosRESUMO
Abstract Background Macrophages are a functionally heterogeneous cell population and depending on microenvironments they polarize in two main groups: M1 and M2. Glutamic acid and glutamate receptors may participate in the regulation of macrophage plasticity. To investigate the role of glutamatergic systems in macrophages physiology, we performed the transfection of mGluR5 cDNAs into RAW-264.7 cells. Results Comparative analysis of modified (RAW-mGluR5 macrophages) and non-modified macrophages (RAW-macrophages) has shown that the RAW-mGluR5 macrophages absorbed more glutamate than control cells and the amount of intracellular glutamate correlated with the expression of excitatory amino acid transporters -2 (EAAT-2). Besides, our results have shown that RAW-mGluR5 macrophages expressed a higher level of peroxisome proliferator-activated receptor γ (PPAR-γ) and secreted more IL-10, high mobility group box 1 proteins (HMGB1) and Galectin-3 than control RAW-macrophages. Conclusions We propose that elevation of intracellular glutamate and expression of mGluR5 may initiate the metabolic rearrangement in macrophages that could contribute to the formation of an immunosuppressive phenotype.
Assuntos
Animais , Camundongos , Receptor de Glutamato Metabotrópico 5/fisiologia , Plasticidade Celular/fisiologia , Macrófagos/fisiologia , Fenótipo , Ensaio de Imunoadsorção Enzimática , Transfecção/métodos , Células Cultivadas , Lipopolissacarídeos , Western Blotting , Interleucina-10/análise , Interleucina-10/metabolismo , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Proteína HMGB1/análise , Proteína HMGB1/metabolismo , Galectina 3/análise , Galectina 3/metabolismo , PPAR alfa/análise , PPAR alfa/metabolismo , Células RAW 264.7 , Óxido Nítrico/metabolismoRESUMO
PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.
Assuntos
Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Mucosa Olfatória/citologia , 5'-Nucleotidase/fisiologia , Animais , Antígenos CD34/fisiologia , Diferenciação Celular/fisiologia , Plasticidade Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Criopreservação , Osso Etmoide/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Mucosa Olfatória/crescimento & desenvolvimento , Coelhos , Antígenos Thy-1/fisiologiaRESUMO
PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.
Assuntos
Animais , Coelhos , Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Mucosa Olfatória/citologia , /fisiologia , /fisiologia , Antígenos Thy-1/fisiologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Criopreservação , Diferenciação Celular/fisiologia , Plasticidade Celular/fisiologia , Proliferação de Células/fisiologia , Osso Etmoide/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Mucosa Olfatória/crescimento & desenvolvimentoRESUMO
BACKGROUND: The endogenous ability to dedifferentiate, re-pattern, and re-differentiate adult cells to repair or replace damaged or missing structures is exclusive to only a few tetrapod species. The Mexican axolotl is one example of these species, having the capacity to regenerate multiple adult structures including their limbs by generating a group of progenitor cells, known as the blastema, which acquire pattern and differentiate into the missing tissues. The formation of a limb regenerate is dependent on cells in the connective tissues that retain memory of their original position in the limb, and use this information to generate the pattern of the missing structure. Observations from recent and historic studies suggest that blastema cells vary in their potential to pattern distal structures during the regeneration process; some cells are plastic and can be reprogrammed to obtain new positional information while others are stable. Our previous studies showed that positional information has temporal and spatial components of variation; early bud (EB) and apical late bud (LB) blastema cells are plastic while basal-LB cells are stable. To identify the potential cellular and molecular basis of this variation, we compared these three cell populations using histological and transcriptional approaches. RESULTS: Histologically, the basal-LB sample showed greater tissue organization than the EB and apical-LB samples. We also observed that cell proliferation was more abundant in EB and apical-LB tissue when compared to basal-LB and mature stump tissue. Lastly, we found that genes associated with cellular differentiation were expressed more highly in the basal-LB samples. CONCLUSIONS: Our results characterize histological and transcriptional differences between EB and apical-LB tissue compared to basal-LB tissue. Combined with our results from a previous study, we hypothesize that the stability of positional information is associated with tissue organization, cell proliferation, and pathways of cellular differentiation.