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Objective • To investigate the effects of estrogen on proliferation, apoptosis and differentiation of bone marrow macrophage (BMM) and the mechanism. Methods • BMMs were isolated from normal C57BL/6J mice and induced to differentiate to osteoclasts in vitro. BMMs in experimental group were administered with 10-8 mol/L exogenous estrogen and antagonist group with both estrogen and 1 μmol/L ICI-182780, an antagonist of estrogen receptor, and control group was designed as well. Five 12-week-old C57BL/6J mice underwent ovariectomy (OVX group) and sham group (n=5) underwent sham surgery. All mice were sacrificed after 3 months to isolate BMM. Proliferation ability of BMM was assessed using Prestoblue, TUNEL assay was performed to detect apoptosis in each group. Caspase 3 and caspase 8 were detected by Western blotting. Quantitative real time PCR was used to detect tartrate-resistant acid phosphatase (Trap) and cathepsin K (Ctsk) mRNA levels during osteoclastogenesis. TRAP staining of osteoclasts showed osteoclastogenesis ability. In addition, the downstream moleculars activated by receptor activator for nuclear factor-κB ligand (RANKL) in BMM were detected by Western blotting. Results • BMM multiplication ability was attenuated in experiment group compared with control group and it was stronger in OVX group than that in sham group. TUNEL assay showed that the apoptotic BMM in experimental group were more than those in control group and caspase 3 and caspase 8 expression were consisted with the results of TUNEL assay. PCR analysis showed that Trap and Ctsk mRNA levels significantly decreased in experiment group compared with control group. The mRNAs increased in OVX group in contrast to sham group. TRAP staining of osteoclasts and quantitative analysis showed that osteoclasts in experiment group were less than those in control group and osteoclasts in OVX group were more than those in sham group. The effects of estrogen on proliferation, apoptosis and differentiation of BMM were blocked by antagonist of estrogen receptor. Western blotting showed that the phosphorylation of IκKα/β, p65 and JNK activated by RANKL were attenuated in experimental group compared with that in control group. Conclusion • Estrogen inhibits proliferation and osteoclastogenesis of BMM, and aggravates their apoptosis through estrogen receptor.
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Objective To investigate the effect of angelica polysaccharide (APS) on bioactivity of bone marrow macrophage (BMM?) and its relationship to hematopoietic regulation, for clarifying the hematonic mechanism of Angelica sinensis. Methods The techniques of hematopoietic progenitor cell culture and BMM? culture in vitro, biological assay of hematopoietic growth factor (HGF) in culture media of BMM?, immunocytochemistry, and nucleic acid in situ hybridization were used. Results The culture supernatant of BMM? induced by APS can enhance the CFU-Mix, CFU-E, CFU-GM; the expression of erythropoietin (EPO), GM-CSF, IL-3, IL-6 protein in BMM? induced by APS was much stronger than that in the control group at different levels; the expression of EPO mRNA and GM-CSF mRNA in BMM? induced by APS were intensified. Conclusion APS may directly and/or indirectly stimulate the BMM? in hematopoietic inductive microenvironment to accelerate the synthesis and secretion of hematopoietic regulation factors on the basis of gene and protein level, such as EPO, GM-CSF, IL-3, IL-6, which in turn to promote the proliferation and differentiation of CFU-Mix, CFU-E, and CFU-GM.
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Objective:To study the biological functions of bone marrow macrophage (BMM ?) and to establish the methodology of separation, purification, cultivation and identification of BMM ?.Methods:Using the techniques of anchorage-dependent culture of separated rat bone marrow cell (rBMC) in DMEM culture media (contain 20% horse serum,20% (v/v)L 929 conditioned media) in vitro,a lot of purified anchor cells were obtained, and these cells were identified with specifically biological marker of macrophage, such as 1, morphologicalobservation:invert phase contrast microscopy, light and electron microscopy;2, enzyme cytochemistry:acid phosphatase (ACP),?-acetic acid naphthol esterase (?-ANE);3,phagocytic experiment:phagocytosis of chicken erythrocytes and prepared Chinese ink;4,immunocytochemistry: surface specific antigen of macrophage ( CD 68 stain).Results:The cells were purified having functional satisfactory macrophage according to morphological observation,enzyme cytochemistry, phagocytic experiment and immunocytochemistry.Conclusion:This is a simple and easy method for separation, purification, cultivation and identification of rat marrow macrophage.