1.Increased levels of multiple forms of dihydrofolate reductase in peripheral blood leucocytes of cancer patients receiving haematopoietic colony-stimulating factors: interim analysis.
M Perwaiz IQBAL ; Ikram A BURNEY ; Fakhra SULTANA ; Naseema MEHBOOBALI ; Tariq SIDDIQUI
Experimental & Molecular Medicine 2000;32(2):84-87
The precise mechanism whereby granulocytes proliferate when haematopoietic colony stimulating factors (CSFs) are used in neutropenic cancer patients is poorly understood. The purpose of this study was to investigate whether these cytokines bring about leucocyte proliferation by increasing the levels of multiple forms of dihydrofolate reductase (DHFR). Blood samples were collected from 36 cancer patients (25 males and 11 females) with chemotherapy-induced neutropenia. One sample of blood from each patient was obtained before therapy either with CSF, such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) or with placebo, and another one at the time of resolution of neutropenia. Peripheral blood leucocytes in these blood samples were counted, separated and lysed. From lysates, cytoplasmic samples were prepared and analyzed for active DHFR by a methotrexate-binding assay and for total immunoreactive DHFR by an enzyme linked immunosorbent assay. The increase in total leucocyte count (TLC) was most prominent (P < 0.005) in the CSF group and less so (P < 0.05) in the placebo group. The mean +/- SD concentration values of active DHFR before and after stimulation with GM-CSF found were to be 0.34 +/- 0.4 ng/mg protein and 0.99 +/- 0.82 ng/mg protein, respectively, and in the group treated with G-CSF, 0.24 +/- 0.32 ng/mg protein and 1.18 +/- 2.4 ng/mg protein, respectively. This increase in active DHFR after stimulation with CSF was statistically significant (P <0.05). Similarly, concentration values of immunoreactive but nonfunctional form of DHFR (IRE) were 110 +/- 97 ng/mg protein and 605 +/- 475 ng/mg protein before and after stimulation with GM-CSF, and 115 +/- 165 ng/mg protein and 1,054 +/- 1,095 ng/ mg protein before and after stimulation with G-CSF. This increase in concentration of IRE after stimulation with GM-CSF or G-CSF was statistically significant (P < 0.005). In the control group, there was an increase in the concentration of both active DHFR and IRE after treatment with placebo. However, this was not statistically significant. Resolution of neutropenia was quicker in the groups treated with CSF compared to the control group. Results of this study indicate that colony stimulating factors (G-CSF and GM-CSF) induce white cell proliferation by increasing the levels of multiple forms of DHFR.
Adolescence
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Adult
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Cell Division/drug effects
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Child
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Female
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Granulocyte Colony-Stimulating Factor/therapeutic use
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Granulocyte Colony-Stimulating Factor/pharmacology*
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Granulocyte Colony-Stimulating Factor/adverse effects
;
Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use
;
Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology*
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Granulocyte-Macrophage Colony-Stimulating Factor/adverse effects
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Human
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Isoenzymes/metabolism
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Isoenzymes/biosynthesis
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Leukocyte Count
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Leukocytes/pathology
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Leukocytes/enzymology
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Leukocytes/drug effects
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Male
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Middle Age
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Neoplasms/enzymology
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Neoplasms/drug therapy
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Neoplasms/blood*
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Neutropenia/metabolism*
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Neutropenia/chemically induce
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Neutropenia/blood
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Tetrahydrofolate Dehydrogenase/metabolism*
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Tetrahydrofolate Dehydrogenase/biosynthesis
2.Modulation of expression of human GM-CSF and GM-CSFRalpha by total saponins of Panax ginseng.
Sha-Li WANG ; Di CHEN ; Ya-Ping WANG ; Yong-Gang LIU ; Rong JIANG
Acta Physiologica Sinica 2003;55(4):487-492
The purpose of the present study was to investigate the biological mechanism for modulating granulocytopoiesis by Panax ginseng. The techniques of culture of hematopoietic progenitor cells and hematopoietic stromal cells in vitro, biological assay of hematopoietic growth factor (HGF), immunocytochemistry, in situ hybridization of nucleic acid, immunoprecipitation and Western blot were used to explore the effect of total saponins of Panax ginseng (TSPG) on the expression of human granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-macrophage colony stimulating factor receptor alpha (GM-CSFRalpha). The results indicated that (1) bone marrow stromal cell (BMSC), thymocyte (TC), splenocyte (SC), endothelial cells (EC), and monocyte (MO) conditioned media prepared with TSPG (50 microg/ml) could significantly enhance the proliferation of CFU-GM; (2) the expressions of GM-CSF in protein and mRNA level in BMSC, TC, SC, EC and MO induced by TSPG (50 microg/ml) were much higher than that of the control; (3) the expression of GM-CSFRalpha protein in hematopoietic cells induced by TSPG (50 microg/ml) was stronger than that of the control; (4) TSPG (50 microg/ml) could stimulate the transient tyrosine phosphorylation of GM-CSFR and Shc protein. We speculate that TSPG may directly and/or indirectly promote the stromal cells and lymphocytes to produce GM-CSF and other cytokine and induce bone marrow hematopoietic cells to express GM-CSF receptors (GM-CSFRalpha), leading to the regulation of the GM-CSFR-mediated signals transduction pathway and the proliferation of human CFU-GM.
Bone Marrow Cells
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cytology
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metabolism
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Cells, Cultured
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Granulocyte-Macrophage Colony-Stimulating Factor
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metabolism
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Hematopoietic Stem Cells
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cytology
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metabolism
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Humans
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Panax
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chemistry
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Receptors, Granulocyte-Macrophage Colony-Stimulating Factor
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metabolism
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Saponins
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isolation & purification
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pharmacology
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Signal Transduction
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Stromal Cells
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cytology
;
metabolism
3.Expression of G-CSF and GM-CSF receptors on CD34 positive cells in aplastic anemia and myelodysplastic syndrome patients and its significance.
Hong-Zhi XU ; Ai LI ; Yuan YU ; Jian-Feng LI ; Xin LIU ; Xiang-Hua WANG ; Xin WANG ; Gong-Li XU
Journal of Experimental Hematology 2008;16(6):1308-1311
This study was aimed to detect the ratio of CD34+ cells in bone marrow mononuclear cells (BMMNCs) and the expression rate of G(M)-CSFR on CD34+ cells in bone marrow of the patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). The ratio of CD34+ cells in BMMNCs and the expression rate of G(M)-CSFR on cells of 27 AA patients, 45 MDS patients and 20 controls were detected by flow cytometry (FCM). The results showed that the ratio of CD34+ cells in BMMNCs of AA patients reduced and was significantly different from controls (p<0.05), the ratio of CD34+ cells in MDS patients elevated and was significantly different from controls (p<0.05). Compared with controls and MDS-RA patients, the ratio of CD34+ cells in MDS-RAEB patients significantly elevated (p<0.05), but there was no significant difference between MDS-RA patients and controls (p>0.05). The ratio of CD34+ cells in MDS-RA patients was significantly higher than that in AA patients (p<0.05). There was no significant difference in expression rate of G-CSFR on CD34+ cells between AA patients and controls, MDS patients and controls, AA patients and MDS patients, MDS-RA patients and MDS-RAEB patients (p>0.05). The expression rate of GM-CSFR in MDS patients was significantly higher than that in AA patients and controls (p<0.05), but there was no significant difference between AA patients and controls, MDS-RA patients and MDS-RAEB patients (p>0.05). In AA patients, the ratio of CD34+ cells in BMMNCs was less than 0.1% accounts for 6/8 SAA patients, compared with 2/19 in CAA (p<0.05). There was no correlation between the expression rate of either G-CSFR or GM-CSFR and neutrophil count at diagnosis (r=0.058 and r=0.044). In MDS patients, there was no correlation between bone marrow CD34+ cells ratio and peripheral neutrophil count at diagnosis (r=-0.335). And there was no correlation between the expression of either G-CSFR or GM-CSFR and neutrophil count on diagnosis (r=0.064 and r=0.051). It is concluded the detection of CD34+ cells and their surface expression rate of G(M)-CSFR in AA and MDS is useful in diagnosis and differential diagnosis of these two diseases.
Adult
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Anemia, Aplastic
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metabolism
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Antigens, CD34
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immunology
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Bone Marrow Cells
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cytology
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immunology
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metabolism
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Case-Control Studies
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Female
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Flow Cytometry
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Humans
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Male
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Middle Aged
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Myelodysplastic Syndromes
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metabolism
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Receptors, Granulocyte Colony-Stimulating Factor
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metabolism
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Receptors, Granulocyte-Macrophage Colony-Stimulating Factor
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metabolism
4.Influence of MicroRNA-382 on Biological Properties of Human Umbilical Cord-Derived Mesenchymal Stem Cells.
Jun-Jie CUI ; Ying CHI ; Xin YANG ; Yu-Yan SHEN ; Zhao WANG ; Su-Dong ZHANG ; Li-Ning ZHANG ; Li LIU ; Shi-Hong LU ; Ming-Zhe HAN ; Si-Zhou FENG
Journal of Experimental Hematology 2016;24(3):852-857
OBJECTIVETo investigate the effect of microRNA-382 (miR-382) on the biological properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSC).
METHODSThe mimics and inhibitor of miR-382 were transfected into hUC-MSC with lipo2000. Inverted microscopy was used to observe the morphology change of hUC-MSC. The proliferation of hUC-MSC was detected by CCK-8. Oil red O and alizarin red staining were applied to assess the adipogenic and osteogenic differentiation of hUC-MSC. Cetylpyridinium chloride was used to the quantitative analysis of osteogenic differentiation. The expression of Runx2 and some cytokines were detected by RT-PCR.
RESULTSmiR-382 did not influence the morphology, proliferation and adipogenic differentiation of hUC-MSC miR-382 inhibited the expression of Runx2, thus could inhibit the osteogenesis of hUC-MSC, being confirmed by alizarin red stain; miR-382 could influence the expression of key cytokines secreted from hUC-MSC, such as IL-6, IDO1, G-CSF, M-CSF, GM-CSF.
CONCLUSIONmiR-382 decreases the expression of Runx2 and inhibites the osteogenesis of hUC-MSC. In addition, it also affects the expression of some key cytokines secreted from hUC-MSC.
Cell Differentiation ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Granulocyte Colony-Stimulating Factor ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Interleukin-6 ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; metabolism ; Osteogenesis ; Transfection ; Umbilical Cord ; cytology
5.Osteoblasts from patients with myelodysplastic syndrome express multiple cytokines and support hematopoietic progenitor cell survival in vitro.
Wen-Ming CHEN ; Zi-Xing CHEN ; Jian-Nong CEN ; Jun HE ; Xue-Li JIAO ; Jin-Lan PAN ; Qiao-Cheng QIU ; Lan DAI ; Dan-Dan LIU
Journal of Experimental Hematology 2008;16(1):78-83
This study was aimed to investigate the biological characteristics of osteoblasts from patients with myelodysplastic syndrome (MDS) and their supportive capacity for hematopoiesis in vitro. A two-dimensional culture system was constructed by using osteoblasts derived from human marrow mesenchymal stem cells (MSC); MSCs were isolated from bone marrow of MDS patients and normal individuals and were cultured; the third passage of MSCs were induced into osteoblasts which were treated with mitomycin C and confluenced into a feeder layer. Ficolled bone marrow mononuclear cells were obtained from normal individuals and seeded into the two-dimensional culture system to culture in vitro without exogenous cytokines. By using colony-forming assay, the ability of the two-dimensional system to culture HPCs was observed. The cytokine expression of osteoblasts from MDS patient bone marrows in mRNA level was detected by RT-PCR and was compared with human osteoblast cell line hFOB1.19. The results showed that the osteoblasts from MDS patients could support short-term survival of GM-CFC in condition without exogenous cytokines, that is, osteoblasts played a crucial role in regulation of HPC growth. The results of RT-PCR clearly demonstrated that the osteoblast cell line hFOB1.19 expressed SCF, IL-6, SDF-1alpha, G-CSF and GM-CSF. The same expression patterns of above cytokines were also seen in osteoblasts derived from BM-MSCs of MDS patients and normal individuals, but these cells did not express GM-CSF. It is concluded that the biological characteristics of osteoblasts from bone marrow of MDS patients are generally not different from those of osteoblasts from normal bone marrow. Both of them can support GM -CFC to form colonies in vitro, it may be associated with expressing important related cytokines by osteoblasts.
Cytokines
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metabolism
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Granulocyte-Macrophage Colony-Stimulating Factor
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metabolism
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Granulocyte-Macrophage Progenitor Cells
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cytology
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Hematopoietic Stem Cells
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cytology
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Humans
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Interleukin-6
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metabolism
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Myelodysplastic Syndromes
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metabolism
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pathology
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Osteoblasts
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metabolism
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physiology
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RNA, Messenger
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metabolism
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Stem Cell Factor
;
metabolism
6.Effect of angiotensin II on cord blood CD34+ cells expansion in vitro.
Cheng, PENG ; Ping, ZOU ; Yanping, MA ; Zongbo, HU
Journal of Huazhong University of Science and Technology (Medical Sciences) 2005;25(1):26-8
In order to investigate the influence of angiotensin II on hematopoietic system, CD34+ cells in cord blood were purified, and the effects of angiotensin II in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin II in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin II concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3 +/- 0.8 times to 7.8 +/- 2.3 times when angiotensin II was added in the presence of SCF+G-CSF+GM-CSF+IL3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1 +/- 1.8 times to 9. 2 +/- 2.3 times with angiotensin II in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin II could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin II had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.
Angiotensin II/*pharmacology
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Antigens, CD34/*metabolism
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Cells, Cultured
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Colony-Forming Units Assay
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Fetal Blood/*cytology
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Granulocyte-Macrophage Colony-Stimulating Factor
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Hematopoietic Stem Cells/*cytology
7.Viperin Deficiency Promotes Polarization of Macrophages and Secretion of M1 and M2 Cytokines.
John EOM ; Jihye YOO ; Jeong Jin KIM ; Jae Bong LEE ; Wanho CHOI ; Chae Gyu PARK ; Jun Young SEO
Immune Network 2018;18(4):e32-
Viperin is a multifunctional protein that was first identified in human primary macrophages treated with interferon-γ and in human fibroblasts infected with human cytomegalovirus. This protein plays a role as an anti-viral protein and a regulator of cell signaling pathways or cellular metabolism when induced in a variety of cells such as fibroblasts, hepatocytes and immune cells including T cells and dendritic cells. However, the role of viperin in macrophages is unknown. Here, we show that viperin is basally expressed in murine bone marrow cells including monocytes. Its expression is maintained in bone marrow monocyte-derived macrophages (BMDMs) depending on macrophage colony-stimulating factor (M-CSF) treatment but not on granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. In wild type (WT) and viperin knockout (KO) BMDMs differentiated with M-CSF or G-MCSF, there are little differences at the gene expression levels of M1 and M2 macrophage markers such as inducible nitric oxide synthase (iNOS) and arginase-1, and cytokines such as IL-6 and IL-10, indicating that viperin expression in BMDMs does not affect the basal gene expression of macrophage markers and cytokines. However, when BMDMs are completely polarized, the levels of expression of macrophage markers and secretion of cytokines in viperin KO M1 and M2 macrophages are significantly higher than those in WT M1 and M2 macrophages. The data suggest that viperin plays a role as a regulator in polarization of macrophages and secretion of M1 and M2 cytokines.
Bone Marrow
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Bone Marrow Cells
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Cytokines*
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Cytomegalovirus
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Dendritic Cells
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Fibroblasts
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Gene Expression
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Granulocyte-Macrophage Colony-Stimulating Factor
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Hepatocytes
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Humans
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Interleukin-10
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Interleukin-6
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Macrophage Colony-Stimulating Factor
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Macrophages*
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Metabolism
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Monocytes
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Nitric Oxide Synthase Type II
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T-Lymphocytes
8.Isolation and characterization of human placenta derived adherent cells and their hematopoietic growth factor expression.
Jin HE ; Yi ZHANG ; Xiao-xia JIANG ; Gang LIU ; Yuan-lin LIU ; He-lian LI ; Ning MAO
Chinese Journal of Hematology 2003;24(12):652-654
OBJECTIVETo isolate and culture human placenta derived adherent cells (hPDAC) and assay their hematopoietic growth factor expression.
METHODSBy enzyme digestion, hPDAC were isolated from human placenta tissue and cultured, and their biological characteristics were studied. The hematopoietic growth factor (HGF) mRNA expression of hPDAC was assayed by RT-PCR.
RESULTShPDAC was successfully isolated from human placenta tissue, which was further confirmed as mesenchymal stem cell-like cells. HGF including SCF, FL, G-CSF, GM-CSF, M-CSF and IL-6 were expressed in hPDAC.
CONCLUSIONhPDAC could be used as feeder layer for umbilical cord blood CD(34)(+) cells ex vivo expansion.
Cells, Cultured ; Female ; Gene Expression ; Granulocyte Colony-Stimulating Factor ; genetics ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Hematopoietic Cell Growth Factors ; genetics ; Humans ; Interleukin-6 ; genetics ; Macrophage Colony-Stimulating Factor ; genetics ; Membrane Proteins ; genetics ; Placenta ; cytology ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; genetics
9.The effects of burn serum on the erythropoiesis and granulopoiesis in bone marrow in mice.
Yan-hong ZHOU ; Cheng-ji LUO ; Zhao-hua GUO ; Pei-yan KONG ; Zhong-min ZOU
Chinese Journal of Burns 2005;21(3):177-180
OBJECTIVETo observe the effects of burn serum on the erythropoiesis and granulopoiesis in bone marrow in mice, and to explore the possible underlying mechanism.
METHODSMurine bone marrow cell (BMC) strain was prepared routinely and was employed in the establishment of the culture system of colony forming units of erythrocytes, or granulocytes and monocytes. To both sets of culture system normal murine serum (N group) and burn serum, which was collected from the mice with 15% full thickness burn at 12 postburn hours (PBH) and 1, 3, 5, 7 and 10 postburn days (PBD), (burn serum group) was added. In addition, positive control and blank control groups were set accordingly. The stimulating activity of all kinds of sera on the BMCs in the two sets of culture system was determined. The changes in the burn serum concentrations of EPO and GM-CSF were detected by radioimmunoassay, and the data were analyzed by logarithmic linear fitting correlation with the former influence of burn sera on the erythrocytes and granulocytes.
RESULTS(1) Burn sera exhibited obvious stimulation promoting activity on the erythropoiesis and granulopoiesis in BMC, and the activity peaked (384 +/- 60 and 127 +/- 16 CFU) on 1 PBD and decreased thereafter to approach the values found in normal sera group (125 +/- 14 and 34 +/- 20 CFU) on 7 PBD. (2) The EPO content in burn serum was evidently higher than the normal value (P < 0.01) during 12 PBH to 7PBD period. The GM-CSF concentration was obviously higher than the normal value (P < 0.05) at 12 PBH and on 1 PBD. (3) The EPO concentration in burn serum was significantly and logarithmically correlated with the stimulation promoting activity of burn serum on erythropoiesis (r = 0.8570, P = 0.0137). But the GM-CSF concentration in culture with burn serum was not correlated with the stimulation promoting activity of burn serum on granulopoiesis (r = 0.7049, P > 0.05).
CONCLUSIONThe sera harvested from burned mice during early postburn stage exhibited strong stimulation promoting activity on the erythropoiesis and granulopoiesis in bone marrow. The increased EPO level in burn serum might be the important factor contributing strong stimulation action on erythropoiesis, while increased GM-CSF level was not.
Animals ; Bone Marrow ; metabolism ; Burns ; blood ; therapy ; Erythropoiesis ; Erythropoietin ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; metabolism ; Granulocytes ; Hematopoietic Stem Cell Mobilization ; Mice ; Mice, Inbred Strains ; Serum ; chemistry
10.Relationship between changes of bone mineral density and bone marrow pathology in ovariectomized rats.
Lei ZHU ; Xiao-Ying ZHAO ; Xi QIU
Journal of Experimental Hematology 2014;22(3):617-622
This study was purposed to investigate the relationship between sequential changes of bone mineral density and bone marrow hematopoiesis in ovariectomized rats, and explore the correlation between the osteoporosis and the hematopoiesis. Fifty female Sprege-Dawley rats aged 3 months were randomly divided into ovariectomized (OVX) and sham operated (Sham) groups, euthanized after 4, 8, 12, 16 and 20 weeks respectively. The bone mineral density (BMD) of left femur was measured. The right femur distal metaphysical cancellous bone was fixed in 10% formalin to observe the changes of the bone marrow pathology. The function of hematopoietic stem cells (HSC) was evaluated by colony forming assay. The level of granulocyte macrophage-colony stimulating factor (GM-CSF) was measured by the enzyme linked immunosorbent assay (ELISA). The results showed that the BMD of femur in OVX 4 week group not decreased significantly as compared with the Sham 4 group, but the volume of adipose tissue significantly increased. The BMD of femur in OVX 8, 12, 16 and 20 weeks decreased significantly as compared with the corresponding Sham groups (P < 0.05). Meanwhile, the volume of hematopoietic tissue decreased and volume of adipose tissue increased, the megakaryocyte number decreased, the number of osteoclasts and mast cells increased in bone marrow section, as compared with sham operated group (P < 0.05). After rats were ovariectomized for 16 weeks, the GM-CSF level was significantly lower than that of Sham group. After rats were ovariectomized for 12 weeks, the GM-CSF level was obviously lower than that of Sham group (P < 0.05). It is concluded that the bone marrow hematopoiesis function also decreases when the BMD of ovariectomized rats reduces. The Sprege-Dawley OVX rats aged 3 months can be used as an model to study the hypohemopoiesis.
Animals
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Bone Density
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Bone Marrow
;
metabolism
;
Disease Models, Animal
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Female
;
Granulocyte-Macrophage Colony-Stimulating Factor
;
blood
;
Osteoclasts
;
metabolism
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Osteoporosis
;
Ovariectomy
;
Rats
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Rats, Sprague-Dawley