Hematopoiesis is the process of blood cell production which occurs in the bone marrow (BM) microenvironment, which is composed by two anatomically and functionally distinct niches: the endosteal and vascular. The endosteal niche hosts hemopoietic stem cells (HSC) in quiescence /self-renewal and the vascular niche are the HSC in proliferation /differentiation. CTH can be divided into two groups: cells capable of renewing indefinitely (LT - HSC) or repopulating for a short period (CT- CTH . Protein malnutrition (PM) is associated with hematologic abnormalities such as leukopenia, anemia and changes in bone microenvironment. Unpublished data from our group demonstrated that DP causes a decrease in femoral bone mineral density of malnourished animals, it changes bone microarchitecture and decreases LT-HSC number. Cortical and trabecular region of malnourished animals showed higher numbers of osteoclasts, as evidenced by tartarate-resistant acid phosphatase reaction (TRAP). Our hypothesis is that DP increases parathormone (PTH) synthesis, which consequently increases RANKL synthesis and decreases osteoprotegerin (OPG) synthesis in osteoblasts. Consequently, RANKL binds to its receptor on osteoclast precursors and induces osteoclast formation. We will also evaluate M-CSF participation in this process as it is produced by osteoblasts and BM stromal cells. RANKL/RANK ligation occurs in the presence of M-CSF, providing osteoclast precursors differentiation into mature osteoclasts. To test this hypothesis, C57Bl/6J mice strain will be induced to PM low protein-diet. Seric calcium and phosphorus will be quantified by commercial kits and parathyroid hormone will be evaluated by ELISA method. Osteoblasts will be isolated by magnetic depletion and expression of OPG and RANKL proteins will be quantified by "western blotting." Protein expression will be evaluated in osteoblasts obtained ex vivo and in cultured cells in the presence of osteogenic induction medium. M-CSF quantification will be performed by ELISA in the culture supernatant stromal MO long term culture and also in osteoblasts culture.
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