1.Modulation of adhesion molecule expression on T cells in bone marrow after in vivo rhG-CSF application.
Ming-Rui HUO ; Ying-Jun CHANG ; Xiang-Yu ZHAO ; Xiao-Hua LUO ; Xiao-Jun HUANG
Journal of Experimental Hematology 2009;17(5):1316-1320
The aim of study was to investigate the modulation effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on adhesion molecule expression of memory T lymphocyte in the bone marrow grafts. rhG-CSF was administered in 41 donors by subcutaneous injection for 5 consecutive days. Bone marrow grafts were collected on day 4. The percentages of CD4+ and CD8+, and the expressions of CD49d, CD54, CD62L and CD11a on donor T cells of steady state-bone marrow grafts (SS-BM, n=11) and rhG-CSF primed bone marrow (G-BM, n=30) were analyzed by using multi-color flow cytometry. The results indicated that the percentages of CD4+ and CD8+ T cells were significantly lower in G-BM than those in SS-BM (p<0.05). There were no significant differences in the percentages of memory CD4+ and CD8+ T cells between SS-BM and G-BM (p>0.05). The expressions of CD49d on CD4+ and CD8+T cells were significantly lower in G-BM than that in SS-BM (p<0.05). Compared with SS-BM, the expressions of CD54 on CD4+, memory CD4+ T cells and CD8+ T cells were significantly lower in G-BM (p<0.05). The expressions of CD62L on CD4+ and CD8+ T cells and memory T cells were all significantly lower in G-BM (p values were all less than 0.001). The expressions of CD11a on CD4+, memory CD4+ T cells were significantly lower in G-BM than that in SS-BM (p<0.05). There were no significant differences in the expression of CD11a on CD8+, memory CD8+ T cells between SS-BM and G-BM (p>0.05). It is concluded that the expression of cell adhesion molecules on the CD4+ and CD8+ T cells in G-BM is down-regulated after rhG-CSF treatment of healthy donors.
Adolescent
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Adult
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Aged
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Bone Marrow
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Bone Marrow Cells
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immunology
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metabolism
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Bone Marrow Transplantation
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immunology
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methods
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Cell Adhesion Molecules
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metabolism
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Female
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Granulocyte Colony-Stimulating Factor
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pharmacology
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Humans
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Living Donors
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Male
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Middle Aged
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Recombinant Proteins
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T-Lymphocytes
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immunology
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metabolism
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Young Adult
2.Immunomodulatory effects of mesenchymal stem cells derived from the bone marrow in acute leukemia patients.
Zhi-gang ZHAO ; Li SUN ; Xiao-fang WANG ; Yi-zhuo ZHANG ; Yong YU ; Hong-liang YANG ; Ping ZOU
Chinese Journal of Oncology 2011;33(2):105-109
OBJECTIVETo study the immunomodulatory effects and mechanisms of mesenchymal stem cells (MSC) derived from the bone marrow in acute leukemia patients in vitro.
METHODSBone marrow mononuclear cells from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) were obtained and cultured in low serum medium. The immunophenotypes were assessed by FACS and immunol histochemistry. The levels of cytokines were evaluated by enzyme linked immunosorbant assay (ELISA). T-cell suppression ability was evaluated by Transwell chamber assay. Moreover, the immunoregulatory ability of AML- and ALL-derived MSC was detected by mixed lymphocyte culture assay.
RESULTSALL-derived MSC showed a typical fibroblast-like morphology. They were positive for CD29, CD44 and CD105, the positive rate were 98.81%, 99.25% and 90.52%, respectively, while negative for CD31, CD45 and CD34. Moreover, ALL- and AML-derived MSC didn't express HLA-DR and co-stirnulatory molecules (CD40, CD80 and CD86). ALL and AML derived MSC could secret several cytokines, such as TGF-β1 (567.58 ± 52.64 and 357.15 ± 33.52), HGF (647.27 ± 102.54 and 219.67 ± 62.37), IL-6 (59.67 ± 15.69 and 54.35 ± 12.31) and IL-11 (102.58 ± 23.54 and 78.21 ± 9.67), the level of secretion of TGF-β1 and HGF were higher in ALL bone marrow derived MSC than that of in AML bone marrow derived MSC. ALL and AML derived MSC significantly suppressed T lymphocyte proliferation in a dose-dependent manner, the counts per minute (CPM) were (3.58 ± 0.54) × 10(4), (2.87 ± 0.33) × 10(4), (1.78 ± 0.51) × 10(4) and (1.15 ± 0.15) × 10(4) for AML derived MSC, and CPM were (1.96 ± 0.31) × 10(4), (1.57 ± 0.28) × 10(4), (0.91 ± 0.41) × 10(4) and (0.22 ± 0.11) × 10(4) for ALL derived MSC when MSC were 0.5 × 10(4), 1 × 10(4), 2 × 10(4) and 5 × 10(4). In addition, the CPM was (4.01 ± 0.72) × 10(4) in control group. The immunosuppressive ability was different between MSCs derived from AML and ALL. The immunosuppressive effect of ALL derived MSC could be reversed by anti-TGF-β1 and anti-HGF antibody.
CONCLUSIONALL-derived MSC show immunoregulatory effect in vitro and this effect is achieved through cytokines. But MSCs derived from AML display abnormal changes in T-cell suppression ability.
Bone Marrow ; immunology ; Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cytokines ; pharmacology ; Humans ; Immunophenotyping ; Interleukin-11 ; metabolism ; Interleukin-6 ; metabolism ; Leukemia, Myeloid, Acute ; immunology ; Mesenchymal Stromal Cells ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; Transforming Growth Factor beta1 ; metabolism
3.Comparison between CD271 and CD133 used for immunomagnetic positive sorting enriching in mesenchymal stem cells from bone marrow.
Wei LIN ; Xue-Mei TANG ; Yuan KONG ; Hui WANG ; Kai-Yan LIU
Journal of Experimental Hematology 2008;16(2):333-338
This study was aimed to find a better method to isolate and enrich mesenchymal stem cells (MSCs)from bone marrow between CD271 (low affinity nerve growth factor receptor, LNGFR) and CD133 used for immunomagnetic positive selections through comparison of characteristics of MSCs isolated by these two agents. CD271+ and CD133+ cells were isolated from bone marrow and their colony forming unit-fibroblast (CFU-F) efficiency and proliferative capacity were assessed. Cell surface phenotype, adipogenic and osteogenic inductions were also assayed on the cells (after passage 3) isolated by both methods. The results showed that the purities of immunomagnetically selected CD271+ and CD133+ cells were (89.50+/-0.98)% and (88.03+/-3.06)% respectively. The CFU-F median frequency of CD271+ cells was 3 times as high as that of CD133+ cells, no CFU-F was observed in CD271- cells, while a few CFU-F was found in the CD133- cells. Phenotype of cells (after passage 3) isolated by the two methods was same, that is CD34-, CD14-, CD45-, CD90+, CD29+, CD44+, CD105+, CD73+. CD271+ cells possessed faster proliferation and stronger osteogenic and adipogenic differentiation potential than that of CD133+ cells. It is concluded that as compared with CD133 positive selection, CD271 positive selection is a better method for isolating and enriching mesenchymal stem cells from bone marrow.
AC133 Antigen
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Antigens, CD
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immunology
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metabolism
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Bone Marrow Cells
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cytology
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Glycoproteins
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immunology
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metabolism
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Humans
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Immunomagnetic Separation
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methods
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Mesenchymal Stromal Cells
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cytology
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Nerve Tissue Proteins
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immunology
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metabolism
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Peptides
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immunology
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metabolism
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Receptors, Nerve Growth Factor
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immunology
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metabolism
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Stem Cells
4.The effect of MSC on cytokine production by naive T cell differentiated in vitro.
Jian-gang JIN ; Jiang-wei HU ; Hong-mei NING ; Kai FENG ; Hu CHEN
Chinese Journal of Hematology 2005;26(6):339-341
OBJECTIVETo investigate the effect of mesenchymal stem cell (MSC) on naive T cell and to explore its mechanism of immunoregulation.
METHODSAfter culturing for 3 passages, MSC was incubated with naive T cells differentiated from cord blood CD34(+) cells in vitro. Variance of cytokine produced by naive T cell in culture supernatant was analyzed by enzyme-linked immunoassays.
RESULTSOn the 7th day of co-culture, a mild proliferation of T cells in the co-culture group was observed: (9.15 +/- 0.68) x 10(5)/well in MSCs + naive T cells group versus (4.87 +/- 1.33) x 10(5)/well in naive T cells alone group (P < 0.05). IFN-gamma production was lower in MSCs + naive T cells group than that in naive T cells alone group: (1.147 +/- 0.181) pg/ml versus (4.897 +/- 0.189) pg/ml (P < 0.05), but IL-2 production was higher in the co-culture group: (16.141 +/- 2.729) pg/ml versus (2.551 +/- 0.460) pg/ml (P < 0.05). Neither IL-4 nor IL-10 were detected.
CONCLUSIONSMSC have allogeneic effect on naive T cell, but may suppress alloreactive T lymphocyte and reduce the incidence of GVHD partly by decreased IFN-gamma production. The result may provide new clues for explaining immunoregulatory mechanism of MSC.
Bone Marrow Cells ; immunology ; Cell Communication ; Cells, Cultured ; Coculture Techniques ; Humans ; Interferon-gamma ; metabolism ; Interleukins ; metabolism ; Mesenchymal Stromal Cells ; immunology ; T-Lymphocytes ; metabolism
5.Abnormal polarization of T lymphocyte induces negative hematopoietic regulation in myelodysplastic syndromes.
Ling-yun WU ; Xiao LI ; Chun-kang CHANG ; Shao-xu YING ; Quan PU
Chinese Journal of Hematology 2007;28(8):549-554
OBJECTIVETo explore polarization of T lymphocyte and its relationship with apoptosis of marrow cells in patients with myelodysplastic syndromes (MDS).
METHODSMeasurements of Th1, Th2, Tc1, Tc2 subsets in bone marrows from 34 patients with MDS and 13 normal controls were performed by flow cytometry. INF-gamma and TNF-alpha in marrow serum were determined by ELISA (Enzyme-linked immunosorbent assay). Apoptosis index of marrow cells was detected by TUNEL (TdT-mediated dUTP nick end labeling). Correlations between Th1, Th2, Tc1, Tc2 subsets and INF-gamma, TNF-alpha levels as well as apoptosis index were analyzed, and relationship between TNF-alpha, INF-gamma levels and apoptosis index was also investigated.
RESULTS(1) The percentage of Th1 cells [(10.1 +/- 1.6)%], Tc1 cells [(24.0 +/- 3.6)%] and Tc1/Tc2 ratio (50.0 +/- 11.1) was significantly increased in patients with MDS than in normal controls [(4.0 +/- 0.5)%, (5.8 +/- 0.6)% and 13.4 +/- 2.7, respectively]. Levels of INF-gamma [(58.6 +/- 21.7) microg/L] and TNF-alpha [(15.7 +/- 3.8) microg/L] in marrow serum of MDS patients was markedly elevated compared to normal controls [0 and (0.3 +/- 0.2) microg/L, respectively]. An increased apoptosis index of nucleated cells was observed in MDS patients [(7.8 +/- 1.5)%] as compared to controls [(2.1 +/- 0.3)%, P < 0.05]. The Th1 cell percentage showed a positive correlation with the levels of INF-gamma and TNF-alpha (r = 0.38, P < 0.05 and r = 0.39, P < 0.05, respectively), and with apoptotic index of nucleated marrow cells in MDS patients (r = 0.33, P < 0.05). Furthermore, a positive correlation was observed between INF-gamma, TNF-alpha levels and apoptotic index of marrow cells (r = 0.74, P < 0.01 and r = 0.73, P < 0.01, respectively). (2) Th1, Tc1 cells and Tc1/Tc2 ratio in MDS-RCMD patients was markedly elevated (P < 0.01) but did not in RCMD-RS, RAEB-1 and RAEB-2 patients as compared to normal controls. (3) An elevation in the percentages of Th1, Tcl and Tc1/ Tc2 ratio was detected in patients with IPSS lower-risk but did not in higher-risk group as compared to controls. (4) Increased Th1 and Tc1 percentages and Th1/Th2 and Tc1/Tc2 ratios were observed in RCMD patients with normal karyotype, but did not in those with abnormal karyotype. Conclusions Th1/Th2 and Tc1/Tc2 in bone marrow of MDS patients were unbalanced, polarizing to type I reaction especially in patients with RCMD subtype, IPSS lower-risk and normal karyotype. The increased Th1 cells in bone marrow may account for the increased apoptosis of nucleated marrow cells in MDS, through proapoptotic cytokines such as INF-gamma and TNF-alpha.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Apoptosis ; immunology ; Bone Marrow ; immunology ; metabolism ; pathology ; Female ; Hematopoietic System ; immunology ; Humans ; Interferon-gamma ; metabolism ; Male ; Middle Aged ; Myelodysplastic Syndromes ; immunology ; T-Lymphocytes ; immunology ; Tumor Necrosis Factor-alpha ; metabolism
6.Effect of human bone marrow mesenchymal stem cells on T lymphocyte killing K562 cells.
Zhi-Gang SI ; Hai BAI ; Cun-Bang WANG ; Zhi-Wen XUE ; Qian WANG ; Tao WU ; Xiao-Liang YANG
Journal of Experimental Hematology 2007;15(6):1216-1219
The objective of this study was to investigate the effect of human bone marrow mesenchymal stem cells (MSCs) on T lymphocyte killing K562 cells. MSCs were isolated from bone marrow and cultured, T cells were harvested by using nylon column method from peripheral blood. The T cells were co-cultured with MSCs, the phenotype expressions of T cell subsets were detected by flow cytometry. Killing effects of T cells (culture alone and co-culture with MSCs) on K562 cells were detected by LDH, expressions of IFN-gamma and IL-4 were detected by ELISA. The results showed that after T cells were co-cultured with MSCs for three days, the proportion of CD4+ and CD4+CD25+ T cells raised significantly (p<0.05) as compared with group of culture alone, but the proportion of CD8+ T cell were not significantly changed (p>0.05). In group of T cells co-cultured with MSCs, killing effects of T cells on K562 cells weakened, at the same time, expression of IFN-gamma decreased while expression of IL-4 increased. It is concluded that the MSCs weaken killing effects of T cells on K562 cells, which associates with increase of CD4+CD25+ T cell subsets and changes of IFN-gamma and IL-4 levels.
Bone Marrow Cells
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cytology
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CD4 Antigens
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immunology
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Coculture Techniques
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Humans
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Interferon-gamma
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metabolism
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Interleukin-2 Receptor alpha Subunit
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immunology
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Interleukin-4
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metabolism
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K562 Cells
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Mesenchymal Stromal Cells
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cytology
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immunology
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T-Lymphocyte Subsets
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immunology
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metabolism
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T-Lymphocytes
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cytology
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immunology
7.Biological features of dendritic cells derived from chronic myeloid leukemia cells in vitro.
Xiang-min TONG ; Jie JIN ; Wen-bin QIAN ; Hai-tao MENG ; Yong-quan XUE
Journal of Zhejiang University. Medical sciences 2005;34(4):348-357
OBJECTIVETo induce primary chronic myeloid leukemia (CML) cells into dendritic cells (DCs).
METHODSBone marrow mononuclear cells (MNCs) were isolated from 13 CML patients and peripheral blood MNCs from 5 healthy donors. The isolated MNCs were co-cultured with rhGM-CSF 1,000 U/ml, rhIL- 4,500 U/ml and TNF-alpha 50 U/ml for 10 days. The morphological features were observed by Wright's staining,inverted microscope and electron microscope. CD(80), CD(86), CD(83), CD(1a) and HLA-DR expression were assayed by flow cytometry, cytogenetic analysis was performed by fluorescence in-situ hybridization(FISH). The concentration of IL-12 was measured by ELISA and the function of antigen presenting was tested by mixed lymphocyte reaction (MLR).
RESULTAfter being cultured with cytokines, the typical dendritic appearance with delicate membrane projections was observed. The CD(80), CD(86), CD(83), CD(1a) and HLA-DR markers and capacity of stimulating allogeneic T cells were upregulated significantly. FISH confirmed that the DCs were generated from leukemic origin and CML DCs could secrete higher level of IL-12 than CML MNCs. There were no differences in morphology and immunophenotype expression between DCs derived from CML and those from normal individuals. However, DCs from CML patients displayed weaker activity than that of normal individuals when tested in MLR.
CONCLUSIONCML cells could be induced into leukemia-DCs by co-culture with cytokines.
Bone Marrow Cells ; immunology ; pathology ; Cell Differentiation ; Dendritic Cells ; cytology ; immunology ; Humans ; Interleukin-12 ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; pathology ; Tumor Cells, Cultured
8.Growth, immunophenotype and interleukin-6 level of bone marrow stromal cells in patients with multiple myeloma and acute leukemia.
Jun-Ling ZHUANG ; Xuan WANG ; Jie-Ping ZHANG ; Yong-Ji WU
Journal of Experimental Hematology 2006;14(4):700-703
The aim of this study was to investigate the growth, immunophenotype and interleukin-6 (IL-6) level of bone marrow stromal cells (BMSC) in patients with acute leukemia (AL) and multiple myeloma (MM). BMSC was cultured by wall-adhesion method and the growth of BMSC was observed. The immunophenotype and cell cycle of BMSC were detected by flow cytometry. The level of interleukin 6 (IL-6) in BMSC culture system was detected by ELISA. The results showed that the primary (17.3 +/- 7.8 days) and continuous (10.3 +/- 3.5 days) growth cycle of BMSC in patients with AL were significantly shorter than those in patients with MM (26.5 +/- 6.3 and 16.5 +/- 4.1 days respectively), and shorter than those in normal controls (25.8 +/- 6.3 and 17.5 +/- 2.4 days) respectively. Similarly, S + G2% (17.4 +/- 3.6%) of BMSC in patients with AL was significantly higher than those in patients with MM (8.5 +/- 2.2%) and in normal controls (8.9 +/- 2.3%). All of the three groups showed positive antigen expressions with CD29 and CD44 were 100%, while CD138, CD34, CD54, CD56 positive were not expressed and CD106 was partially expressed positive. The supernatant IL-6 level of BMSC system in MM patients (1288.5 +/- 736.7 pg/ml) was significantly higher than those in AL patients (859.3 +/- 203.1 pg/ml) and normal controls (850.9 +/- 129.5 pg/ml). It is concluded that the growth, S + G2% of cell cycle and IL-6 level of BMSC in patients with MM, AL and normal control are significantly different, whereas the antigen expressions are similar.
Acute Disease
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Bone Marrow Cells
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immunology
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metabolism
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pathology
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Cell Proliferation
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Humans
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Hyaluronan Receptors
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analysis
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Immunophenotyping
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Integrin beta1
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analysis
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Interleukin-6
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analysis
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Leukemia
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immunology
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metabolism
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pathology
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Multiple Myeloma
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immunology
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metabolism
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pathology
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Stromal Cells
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immunology
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metabolism
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pathology
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Tumor Cells, Cultured
9.Hemangioblastic characteristics of fetal bone marrow-derived Flk1+ CD34- cells.
Hong GUO ; Zhi-gang ZHAO ; Jie-wen LIU ; Hui-shu CHEN ; Chun-hua ZHAO
Acta Academiae Medicinae Sinicae 2002;24(6):606-610
OBJECTIVETo investigate whether fetal bone marrow stromal cells have hemangioblastic characteristics.
METHODSHuman fetal bone marrow stromal cells (hfMSCs) were isolated and cultured. Immunophenotypes of hfMSCs were tested by FACS. hfMSCs seeded in the matrigel were induced with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in vitro. Vascularization and hematopoiesis were detected with immunohistochemistry and electron microscope.
RESULTSThe typical properties of this CD34- stromal cell population were that 99% cells expressed Flk1 (vascular endothelial cell growth factor receptor 2) and tube structure was formed. In the process of induction, hfMSCs could give rise to CD34+ round cells.
CONCLUSIONSWe have demonstrated that fetal bone marrow stroma-derived Flk1+ CD34- cells could differentiate into vascular endothelial cells and hematopoietic cells, indicating that fetal bone marrow stroma-derived Flk1+ CD34- cells have hemangioblastic characteristics.
Antigens, CD34 ; immunology ; Bone Marrow Cells ; cytology ; immunology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Fetus ; Fibroblast Growth Factor 2 ; metabolism ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; immunology ; metabolism ; Humans ; Immunophenotyping ; Stromal Cells ; cytology ; immunology ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; physiology
10.Expression and clinical significance of Notch1 on the membrane of bone marrow CD38(+)CD138(+) plasma cells in the patients with multiple myeloma.
Yi-ran ZHAO ; Rong FU ; Jing GUAN ; Shan GAO ; Hui LIU ; Er-bao RUAN ; Wen QU ; Yong LIANG ; Guo-jin WANG ; Xiao-ming WANG ; Hong LIU ; Yu-hong WU ; Jia SONG ; Hua-quan WANG ; Li-min XING ; Jun WANG ; Li-juan LI ; Zong-hong SHAO
Chinese Journal of Hematology 2012;33(4):274-277
OBJECTIVETo investigate the expression of Notch1 on the membrane of bone marrow CD38(+)CD138(+) plasma cells in the patients with multiple myeloma (MM), and explore the importance of Notch signaling pathway in the formation and progression of MM.
METHODSThirty three MM patients and 15 healthy controls were enrolled in this study. The expression of Notch1 on the membrane of bone marrow CD38(+)CD138(+) and CD38(+)CD138(-) plasma cells were analyzed by flow cytometry. The clinical data of MM patients were also analyzed.
RESULTSThe ratio of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients was (60.21 ± 25.06)% which was significantly higher than those of CD38(+)CD138(-) plasma cells of MM patients (39.84 ± 18.94)% (P = 0.000) and controls (38.34 ± 19.39)% (P = 0.004). There was no statistical difference between the two latter groups (P > 0.05). The expression of Notch1 on CD38(+)CD138(+)plasma cells from 24 newly diagnosed MM patients was correlated to the level of malignant plasma cells in there bone marrow (r = 0.914, P = 0.000), serum level of lactate dehydrogenase (LDH) (r = 0.754, P = 0.007), and β(2)-MG(r = 0.716, P = 0.013). The ratio of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients who had renal dysfunction was correlated to their abnormal serum creatinine levels. The expression of Notch1 on CD38(+)CD138(+) plasma cells from 17 MM patients who received VD (bortezamib and dexamethasone) chemotherapy was correlated to the ratio of plasma cell reduction after the first VD chemotherapy (r = 0.842, P = 0.000).
CONCLUSIONThe expression of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients was significantly higher than those of CD38(+)CD138(-) plasma cells of MM patients and controls. Notch1 overexpressed plasma cells were sensitive to the early VD therapy, and correlated to the progression and long term outcome of MM.
ADP-ribosyl Cyclase 1 ; immunology ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; metabolism ; Case-Control Studies ; Cell Count ; Female ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; immunology ; metabolism ; Plasma Cells ; immunology ; metabolism ; Prognosis ; Receptor, Notch1 ; metabolism ; Syndecan-1 ; immunology