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
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Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use
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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.Comparison of Several Optimization Schemes for the Induction and Expansion of Antibody-Mediated High Efficiency CIK (AMHE-CIK) In Vitro.
Xue YIN ; Xin XU ; Yao ZHAO ; Zhan-Ju WANG ; Hai-Ying WANG ; Zhen-Bo HU
Journal of Experimental Hematology 2016;24(1):191-196
OBJECTIVETo compare several schemes of inducing and expanding the antibody-mediated high efficiency CIK (AMHE-CIK) in vitro, so as to find out a method that can acquire a large number of cells capable to kill the tumor cells in a short time.
METHODSPeripheral blood mononuclear cells (PBMNC) from healthy volunteers was isolated and activated with CD3 antibody, then were cultured with the addition of different cytokines (IL-2, IL-4, G-CSF, GM-CSF, IFN-γ, TNF-α) for 14 days in vitro. The morphological changes of cells were observed by light microscopy. Based on the immunophenotypes of cells in each groups analyzed by flow cytometry, the cytokines capable to induce the dendritic cells and killer cells were screened out, respectively. According to different combination of cytokines, the cells were divided 4 groups: control, IL-2, group 1 (componant A included IL-2, IL-4, and GM-CSF. Componant B included IL-2, G-CSF, IFN-γ, and TNF-α), and group 2 (componant A included IL-2, IL-4, and GM-CSF. Componant B included IL-2, IL-4, G-CSF, IFN-γ, and TNF-α). The proliferation and differentiation of CD3(+) CD8(+) and CD3(+) CD56(+) cells were measured by flow cytometry after culture in vitro for 7 days.
RESULTSAfter inducing and expanding in vitro for 7 days, the cell proliferation rate of control group, IL-2 group, group 1 and group 2 were 1.57 ± 0.01, 4.17 ± 0.16, 5 ± 0.47, 7.17 ± 0.24-folds, respectively. The differences between IL-2 group, group 1, group 2 and control group were statistically significant (P < 0.05). The immunophenotype analysis showed that the proportion of CD3(+) CD8(+) induced by each protocol was 13.96 ± 0.23%, 26.33 ± 0.55%, 36.83 ± 0.34% and 35.88 ± 0.16%, respectively. The proportion of CD3(+) CD8(+) in group 1 and 2 was higher than that in IL-2 group (P < 0.05), but the difference between them was not significant (P < 0.05). The proportions of CD3(+) CD56(+) induced by each protocol were 11.03 ± 0.28%, 29.31 ± 0.60%, 39.96 ± 0.38% and 29.33 ± 0.54%, respectively, the proportion of group 1 was higher than that of IL-2 group and group 2 (P < 0.05), but the difference between IL-2 group and group 2 was not significant (P < 0.05).
CONCLUSIONThe group 1 protocol obtained from this study can promote the proliferation of DC-CIK and also increase the proportion of the tumor killing cells (CD3(+) CD8(+) and CD3(+) CD56(+)).
Cell Culture Techniques ; Cells, Cultured ; Culture Media ; chemistry ; Cytokine-Induced Killer Cells ; cytology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunophenotyping ; Interferon-gamma ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-4 ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology
3.Stimulating effect of catechin, an active component of Spatholobus suberectus Dunn, on bioactivity of hematopoietic growth factor.
Dong-xiao WANG ; Ping LIU ; Yi-hong CHEN ; Ruo-yun CHEN ; Dai-hong GUO ; Hao-yang REN ; Meng-li CHEN
Chinese Medical Journal 2008;121(8):752-755
BACKGROUNDHematopoietic growth factor (HGF) is indispensable to hematopoiesis in the body. The proliferation and differentiation of hematopoietic cells must rely on the existence and stimulation of HGF. This study investigated the effect of catechin, an active component extracted from Spatholobus suberectus Dunn (SSD), on bioactivity of granulocyte-macrophage colony-stimulating activity (GM-CSA), burst-promoting activity (BPA) and megakaryocyte colony-stimulating activity (MK-CSA) in spleen condition medium (SPCM) of mice to clarify the hematopoietic mechanism of catechin and SSD.
METHODSSpleen cells of mice were separated and spleen condition medium (SPCM) was prepared from spleen cell culture. Bone marrow cells of mice were separated and cultured in a culture system including 10% (v/v) SPCM (induced by catechin in vivo or ex vivo) for 6 days. Granulocyte-macrophage colony forming units (CFU-GM), erythrocyte burst-colony-forming units (BFU-E) and megakaryocyte colony-forming units (CFU-Meg) formation were employed to assay the effects of different treatment on the bioactivity of GM-CSA, BPA and MK-CSA in SPCM.
RESULTSSPCM induced by 100 mg/L catechin ex vivo could promote the growth of CFU-GM, BFU-E and CFU-Meg, which indicated that catechin could stimulate the production of GM-CSA, BPA and MK-CSA in SPCM. SPCM prepared at the fourth day of spleen cell culture showed the best stimulating activity. The bioactivity of GM-CSA, BPA and MK-CSA in the SPCM prepared after intraperitoneally injecting catechin into mice was also increased. The number of CFU-GM, BFU-E and CFU-Meg gradually increased as the dose of catechin increased and the time of administration prolonged. CFU-GM, BFU-E and CFU-Meg of the high-dose catechin group were significantly higher than those of the control group (P < 0.01) and reached the maximum at the seventh day after administration.
CONCLUSIONSThis study suggests that catechin extracted from the active acetic ether part of Spatholobus suberectus Dunn can regulate hematopoiesis by inducing bioactivity of GM-CSA, BPA and MK-CSA in SPCM of mice. This may be one of the mechanisms for the hematopoietic-supportive effect of catechin and Spatholobus suberectus Dunn.
Animals ; Catechin ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; physiology ; Hematopoiesis ; drug effects ; Interleukin-3 ; physiology ; Mice ; Thrombopoietin ; physiology
4.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
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metabolism
5.Ex vivo generation of functional dendritic cells from mobilized CD34+ hematopoietic stem cells.
Yoo Hong MIN ; Seung Tae LEE ; Kyung Mi CHOI ; So Young CHONG ; Hyun Ok KIM ; Jee Sook HAHN ; Yun Woong KO
Yonsei Medical Journal 1998;39(4):328-338
The ability to generate dendritic cells (DCs) in sizeable numbers has enormous implications for the development of clinically-effective antigen presentation procedures for cancer immunotherapy. We evaluated the generation of immunostimulatory DCs from peripheral blood CD34+ cells collected from healthy donors. CD34+ cells purified from leukapheresis product were seeded at 1 x 10(4) cells/mL in complete medium supplemented with GM-CSF, TNF alpha, IL-4, c-kit ligand, and flt3 ligand (FL). By day 14 of culture in the presence of GM-CSF + TNF alpha, the total cell number increased by 23.4 +/- 5.4-fold compared to the starting number of CD34+ cells. When the c-kit and FL were added to GM-CSF and TNF alpha, the cell number increased by 109.8 +/- 11.2-fold without affecting the immunophenotype of recovered cells. Flow cytometric analysis indicated that cells with the markers of mature dendritic cells, i.e., CD1a +CD14 -HLA-DR+, and CD80+CD86+HLA-DR+, constituted 49.0% +/- 7.5%, and 38.9% +/- 6.5%, respectively. This pattern of expression of surface antigen was unchanged whether the c-kit ligand and/or FL was added. The irradiated CD1a+HLA-DR+ cells recovered from in vitro cultures elicit a vigorous proliferation of allogeneic peripheral blood T-cells, irrespective of cytokine combinations. These findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.
Antigens, CD34/analysis*
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Dendritic Cells/physiology*
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Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology
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HLA-DR Antigens/analysis
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Hematopoietic Stem Cells/physiology*
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Human
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Interleukin-4/pharmacology
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Tumor Necrosis Factor/pharmacology
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.IL-1β promotes the hematopoietic support of human umbilical cord mesenchymal stem cells.
Yue-Ru JI ; Zhou-Xin YANG ; Li-Na LI ; Zhi-Bo HAN ; Ying CHI ; Zhong-Chao HAN
Journal of Experimental Hematology 2013;21(4):1005-1009
This study was aimed to investigate the effect of IL-1β on hematopoietic support of human umbilical cord mesenchymal stem cells (hUC-MSC). 2×10(6) hUC-MSC were seeded in 75 cm(2) flasks, after adherence to wall for 2 h, 10 ng/ml IL-1β was added in hUC-MSC supernatant and cultured for 36 h, then the culture supernatants and cells were harvested. The effect of conditioned medium with/without IL-1β on CD34(+) cell hematopoietic support was observed, mRNA expression changes of hUC-MSC cultured in medium with/without IL-1β were monitored by real time PCR, the differences in hematopoiesis-related factors were detected by ELISA. The results showed that the conditioned culture medium of hUC-MSC with IL-1β enhanced the ability to form colony of CD34(+) cells, especially CFU-G and CFU-GM in vitro; IL-1β promoted the mRNA expression of GM-CSF, G-CSF, IL-6 on MSC; IL-1β also promoted the secretion of GM-CSF, G-CSF, and IL-6 protein from hUC-MSC. It is concluded that IL-1β enhances hematopoietic support capacity especially, capability of MSC to myeloid differentiation.
Cell Differentiation
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Cells, Cultured
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Culture Media
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Granulocyte Colony-Stimulating Factor
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secretion
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Granulocyte-Macrophage Colony-Stimulating Factor
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secretion
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Hematopoietic System
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drug effects
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Humans
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Interleukin-1beta
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pharmacology
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Interleukin-6
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secretion
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Mesenchymal Stromal Cells
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cytology
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drug effects
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secretion
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Umbilical Cord
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cytology
8.In vitro amplification and identification of immature dendritic cells from murine bone marrow.
Chinese Journal of Burns 2003;19(6):332-335
OBJECTIVETo establish a method for in vitro amplification of immature dendritic cells from murine bone marrow, and to identify it with morphological, immunological phenotype determination, and functional examination.
METHODSDendritic cells from murine bone marrow were cultured with different dosage of rmGM-CSF. The suspending cells were examined with scanning electronic microscope, and the non-sensitized T lymphocyte proliferation was observed by mixed lymphocyte reaction.
RESULTSDendritic cells (DC) cultured in lower dosage of rmGM-CSF (Gm(low)DC) exhibited typical characteristics of DCs with high expression of CD11c and low expression of CD40 and I-A/I-E, and non-expression of B7-1 on the surface of the cells. The capacity of Gm(low)DC to stimulate the proliferation of non-sensitized T lymphocyte in vitro was weaker than that of Gm(high)DC.
CONCLUSIONGm(low)DC exhibited typical characteristics of DC, immature in cell phenotype and cell functions, suggesting that our methods of immature DCs culturing was feasible. The dosage of rm GM-CSF has direct relationship with the maturation degree of DC. Generally speaking, mature DC was mainly induced by high dosage of rmGM-CSF, while immature DC by low dosage.
Animals ; Bone Marrow Cells ; physiology ; Burns ; surgery ; Cell Division ; Dendritic Cells ; physiology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; immunology
9.Generation of streptavidin-tagged human-granulocyte macrophage colony-stimulating factor fusion proteins.
Li BAI ; Zhiming HU ; Fei WANG ; Xiaoling XU ; Chang XIA ; Liqin JIN ; Jinlong LI ; Jimin GAO
Journal of Southern Medical University 2012;32(10):1389-1393
OBJECTIVETo obtain streptavidin-tagged human granulocyte-macrophage colony-stimulating factor (SA/hGM-CSF) fusion protein and evaluate its bioactivity .
METHODSPET24a-6His-SA-L-hGM-CSF and PET24a-hGM-CSF-L-SA-6His plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate the fusion proteins. The two fusion proteins were refolded by gradient dialysis after Ni-NTA affinity chromatography and finally purified using DEAE-sepharose FF anion exchange chromatography. MTT method was used to evaluate the effect of SA/hGM-CSF fusion proteins in inducing the proliferation of human erythroleukemia cells (TF-1). The efficiency of the fusion proteins for surface modification of biotinylated MB49 tumor cells was evaluated by flow cytometry.
RESULTSThe recombinant fusion proteins SA-hGM-CSF and hGM-CSF-SA were highly expressed in Rosetta (DE3) at about 20% of the total bacterial proteins, with a purity of about 96% after purification. The two fusion proteins exhibited bifunctional activities, namely the pro-proliferation effect on human erythroleukemia cells (TF-1) and SA-mediated high-affinity binding to biotinylated cell surfaces (with an anchoring modified rate of about 99%).
CONCLUSIONSA/hGM-CSF bi-fusion proteins obtained in this study lays the groundwork for the development of cancer cell vaccines with surface modification by hGM-CSF.
Biomarkers ; Cancer Vaccines ; biosynthesis ; Cell Line, Tumor ; Diterpenes ; pharmacology ; Escherichia coli ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; Humans ; Membrane Fusion Proteins ; biosynthesis ; Plasmids ; Streptavidin ; chemistry
10.Effects of granulocyte-macrophage colony stimulating factor on the repair of vessel intima damaged by balloon.
Xing-Hua ZHANG ; Xiao-Jing MA ; Tong ZHAO
Chinese Medical Journal 2005;118(3):220-225
BACKGROUNDThe dysfunction of vascular endothelial cells plays a key role in starting and facilitating restenosis. The acceleration of intima repair and the recovery of endothelial function would reduce the restenosis rate. This study was undertaken to assess the effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on the repair of damaged iliac arteries.
METHODSTwenty-four male New Zealand white rabbits undergoing primary iliac artery deendothelization were randomly divided into two groups (GM-CSF group and control group). The GM-CSF group received a subcutaneous injection of GM-CSF [10 microg x kg(-1) x d(-1)], and the control group was given a subcutaneous injection of equivalent saline. The iliac arteries of all animals were damaged by balloon after 7 days. The levels of nitric oxide (NO) were detected before, 1 week, 2 weeks and 4 weeks after angioplasty. The repair and hyperplasia of the intima were observed microscopically and the indices of stenosis were evaluated by computerized planimetry after 4 weeks of angioplasty.
RESULTSThe NO levels of the GM-CSF group were higher than those of the control group 2 weeks and 4 weeks after angioplasty [(91.92 +/- 11.57) micromol/L vs. (81.67 +/- 12.18) micromol/L; (97.67 +/- 10.13) micromol/L vs. (83.16 +/- 12.64) micromol/L]. Four weeks after balloon damage, histological examination showed that neointima formation, vascular smooth muscle cells and fibrous tissue of the GM-CSF group were less than those of the control group. The endothelium of the GM-CSF group was more integrated, and stenosis of lumen was slighter than that of the control group. Morphometry showed the lumen area of the GM-CSF group was larger than that of the control group [(1.27 +/- 0.31) mm(2) vs. (0.92 +/- 0.24) mm(2)], the neointimal area and percent of intima hyperplasia were significantly smaller than those of the control group [(0.85 +/- 0.34) mm(2) vs. (1.18 +/- 0.38) mm(2); (40 +/- 7)% vs. (55 +/- 6)%].
CONCLUSIONGM-CSF could facilitate the repair of the intima, reduce neointima formation, better the function of the endothelium, and decrease the rate of restenosis.
Angioplasty, Balloon ; adverse effects ; Animals ; Endothelium, Vascular ; pathology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Hyperplasia ; Iliac Artery ; Male ; Nitric Oxide ; blood ; Rabbits ; Tunica Intima ; drug effects ; pathology