• Title, Summary, Keyword: M-CSF

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Cloning of Bovine Macrophage Colony-stimulating Factor

  • Kim, Tae-Yung;Kim, Cheol-Ho;Lee, Sang-Gil;Kang, Chung-Boo
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.6
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    • pp.892-897
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    • 2005
  • Macrophage colony-stimulating factor (M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocyte lineage. Total and 16 poly (A) mRNA of bovine M-CSF were isolated from healthy bovine peripheral mononuclear cells stimulated by phobol 12-myristste 13-acetate (TPA). The more compatible cultured mononuclear cells were 5${\times}$10/ml for RNA isolation. TPA-activated mononuclear cells increased the level of M-CSF-mRNA more than concanavalin A (Con A) and lipopolysaccharide (LPS). The optimal analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) for14 Macrophage colonystimulating factor (M-CSF) as a growth factor required for bovine M-CSF was denaturation at 94$^{\circ}C$ for 1 minute, annealing at 57$^{\circ}C$ for 1 minute, extension at 72$^{\circ}C$ for 1 minute for 30 cycles. The size of cDNA of bovine M-CSF by RT-PCR was 774 base pairs. A 774 base pairs cDNA encoding bovine M-CSF was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Ligated cDNA was transformed to competent cells and then plasmid isolation and digestion was performed. Molecular cloning and sequencing were performed for cDNA of bovine M-CSF. The size of cloned cDNA of bovine M-CSF was 774base pairs. The homology of base sequence and amino acid sequence was 88% and 86% compared with known human M-CSF, respectively. From a high degree of sequence similarity, the obtained cDNA of bovine M-CSF is thought be a specific gene of bovine M-CSF.

Characterization of Double Transgenic Mice Harboring Both Goat $\beta$-casein/hGH and Goat $\beta$-casein/hG-CSF Hybrid Genes

  • Oh, Keon-Bong;Lee, Chul-Sang
    • Development and Reproduction
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    • v.13 no.3
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    • pp.191-198
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    • 2009
  • In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

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Effects of compressive stress on the expression of M-CSF, IL-$1{\beta}$, RANKL and OPG mRNA in periodontal ligament cells (압박력이 치주인대 세포의 M-CSF, IL-$1{\beta}$, RANKL 및 OPG mRNA 발현에 미치는 영향)

  • Kim, Ji-Woong;Lee, Ki-Soo;Nahm, Jong-Hyun;Kang, Yoon-Goo
    • The korean journal of orthodontics
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    • v.39 no.4
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    • pp.248-256
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    • 2009
  • Objective: The aim of this study was to determine if human PDL cells can produce osteoclastogenic mRNA and examine how compressive stress affects the expression of osteoclastogenic mRNA in human PDL cells. Methods: Human PDL cells were obtained from biscupids extracted for orthodontic treatment. The compressive force was adjusted by increasing the number of cover glasses. PDL cells were subjected to a compressive force of 0.5, 1.0, 2.0, 3.0 or $4.0\;g/cm^2$ for 0.5, 1.5, 6, 24 or 48 hours. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to examine levels of M-CSF, IL-$1{\beta}$, RANKL, OPG mRNA expression. Results: Human PDL cells could produce M-CSF mRNA. Human PDL cells under compressive stress showed increased M-CSF, IL-$1{\beta}$ and RANKL mRNAs expression in a force (up to $2\;g/cm^2$) and time-dependent manner. However, OPG mRNA expression was constant regardless of the level and duration of stress. Conclusions: Continuous compressive stress induced the mRNA expression of osteoclastogenic cytokines including M-CSF, RANKL, IL-$1{\beta}$ in PDL cells. Together with an unchanged OPG mRNA level, these results suggest that compressive stress-induced osteoclastogenesis in vivo is partly controlled by M-CSF, RANKL and IL-$1{\beta}$ expression in PDL cells.

Cultivation of Transgenic Nicotiana tabacum Suspension Cells in Bioreacters for the Production of mGM-CSF

  • Lee, Sang-Yoon;Won Hur;Cho, Gyu-Heon;Kim, Dong-Il
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.6 no.1
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    • pp.72-74
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    • 2001
  • Transgenic Nicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred tank bioreactor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor, 9.0g/L of cells and 2.2ng/mL of mGM-CSF were obtained (11.0g/L and 2.4ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred thank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0g/L due to better mixing by agitation at the higher cell density.

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담배세초현탁배양을 이용한 human granulocyte-macrophage colony stimulating factor의 생산에서 배지 성분이 미치는 영향

  • Lee, Gi-Yong;Lee, Sang-Yun;Myeong, Hyeon-Jong;No, Yun-Suk;Kim, Dong-Il
    • 한국생물공학회:학술대회논문집
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    • pp.325-328
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    • 2002
  • Production of human granulocyte-macrophage colony stimulating factor (hGM-CSF) by Nicotiana tabacum cell suspension culture was studied in Murashige and Skoog (MS) medium with sucrose as a carbon source, ammonium nitrate and potassium nitrate as nitrogen sources, potassium dihydrogen phosphate and sodium dihydrogen phosphate hydrate as phosphate sources, respectively. Optimum concentrations for carbon, nitrogen, phosphate was determined to enhance the production of hGM-CSF. Cell growth was better at high initial sucrose concentration (60 g/L), high initial nitrogen concentration (121.04 mM). Maximum cell density (18.28 g/L) was obtained at 60 g/L of sucrose after 14 days. Cell growth was not so good at low initial sucrose concentration 00 g/L), but the highest hGM-CSF production was obtained at the latter half of exponential phase. hGM-CSF production increased about 3 fold at initial phosphate concentration of 4.96 nM

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A2B Adenosine Receptor Stimulation Down-regulates M-CSF-mediated Osteoclast Proliferation

  • Oh, Yoon Taek;Lee, Na Kyung
    • Biomedical Science Letters
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    • v.23 no.3
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    • pp.194-200
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    • 2017
  • Bone-resorbing osteoclasts play a major role in maintaining bone homeostasis with bone-forming osteoblasts. Although it has been reported that A2B adenosine receptor (A2BAR) regulates osteoclast differentiation, its effects on apoptosis or proliferation of osteoclasts have been less-defined. Here, we demonstrate that A2BAR stimulation regulates macrophage-colony stimulating factor (M-CSF)-mediated osteoclast proliferation. Stimulation with a specific agonist of A2BAR, BAY 60-6583, significantly reduced M-CSF-mediated osteoclast proliferation in a time- and dose-dependent manner. In addition, A2BAR stimulation induced both apoptosis of the cells and cell arrest in the G1 phase with a decrease of cell number in the G2/M phase. Stimulation with BAY 60-6583 inhibited the activation of Akt by M-CSF, whereas M-CSF-induced ERK1/2 activation was not affected. These results suggest that the inhibition of M-CSF-mediated Akt activation by A2BAR stimulation increases apoptotic response of osteoclasts and induces cell cycle arrest in the G1 phase, thus contributing to the down-regulation of osteoclast proliferation.

Anti-inflammatory Effect of Angelicae Gigantis Radix Water Extract on LPS-stimulated Mouse Macrophages (Lipopolysaccharide로 유발된 마우스 대식세포의 염증매개성 Cytokine 생성증가에 대한 참당귀 물추출물의 효능 연구)

  • Han, Hyo-Sang
    • The Korea Journal of Herbology
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    • v.28 no.5
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    • pp.113-119
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    • 2013
  • Objectives : The purpose of this study was to investigate the effects of Angelicae Gigantis Radix Water Extract(AG) on the production of proinflammatory mediators in RAW 264.7 cells stimulated with lipopolysaccharide(LPS). Method : RAW 264.7 cells were cotreated with AG(50 and 100 ug/mL) and lipopolysaccharide(LPS; 1 ug/mL) for 24 hours. After 24 hour treatment, using Bead-based multiplex cytokine assay, concentrations of various cytokines such as interleukin(IL)-6, IL-$1{\beta}$, IL-10, tumor necrosis factor-alpha(TNF-${\alpha}$), granulocyte colony-stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF), interferon inducible protein-10(IP-10), leukemia inhibitory factor(LIF), lipopolysaccharide-induced chemokine(LIX), monocyte chemoattractant protein-1(MCP-1), macrophage colony-stimulating factor(M-CSF), macrophage inflammatory protein(MIP)-$1{\alpha}$, MIP-$1{\beta}$, MIP-2, Regulated on Activation, Normal T cell Expressed and Secreted(RANTES) and vascular endothelial growth factor(VEGF) were measured. Result : AG significantly inhibited LPS-induced production of TNF-${\alpha}$, MIP-$1{\alpha}$, G-CSF, RANTES, IL-10, and M-CSF from LPS-stimulated RAW 264.7 cells at the concentrations of 50 and 100 ug/mL. AG significantly inhibited LPS-induced production of MIP-$1{\beta}$, MIP-2, GM-CSF, and IL-6 from LPS-stimulated RAW 264.7 cells at the concentrations of 50 ug/mL. AG significantly inhibited LPS-induced production of VEGF from LPS-stimulated RAW 264.7 cells at the concentrations of 100 ug/mL. But AG did not show any significant effect on the production of MCP-1, LIF, LIX, IP-10 and IL-$1{\beta}$ from LPS-induced RAW 264.7 cells. Conclusion : These results suggest that AG has anti-inflammatory effect related with its inhibition of proinflammatory mediators such as TNF-${\alpha}$, MIP-$1{\alpha}$, G-CSF, RANTES, IL-10, MIP-$1{\beta}$, MIP-2, GM-CSF, IL-6, VEGF and M-CSF in LPS-induced macrophages.

Biological Activity of Recombinant Human Granulocyte Colony-Stimulating Factor and Isolation of the Somatic Cell Transfected EGFP-hG-CSF Gene (유전자 재조합 인간의 G-CSF의 생리활성과 EGFP-hG-CSF유전자가 도입된 체세포의 분리)

  • Park, Jong-Ju;Min, Kwan-Sik
    • Journal of Life Science
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    • v.18 no.7
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    • pp.912-917
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    • 2008
  • To investigate the biological activity of recombinant human granulocyte colony-stimulating factor (rec-hG-CSF) in mammalian cells, hG-CSF gene was cloned using the eDNA extracted from the human squamous carcinoma cell lines and rec-hG-CSF was produced in CHO cell lines. To analyze the biological activity in vivo, the rec-hG-CSF protein was injected into mice subcutaneously on days 0 and 2. Blood was withdrawn for white blood cell (WBC) determination 5 days after the first injection. WBC values were found to have increased significantly. A pEGFP-mUII-hG-CSF vector was transfected into somatic cell lines isolated from bovine fetal cells. The colony expressing EGFP signals was observed with a confocal microscope. These data suggest that the rec-hG-CSF produced in this study has potent activity in vivo. Thus, the results of this biological activity show that rec-hG-CSF can be enhanced considerably by genetic engineering that affects potential activity, including mutations, which add the oligosaccharide chain and construct double-fusion proteins. A pEGFP-mUII-hG-CSF vector can be utilized for the production of cloned transgenic livestock.

Evaluation of reference value of anti-Glutamic acid decarboxylase antibody for cerebrospinal fluid (뇌척수액에서 항 Glutamic acid decarboxylase 항체검사의 참고치 설정)

  • Park, Min-Ho;Shin, Sun-Young;Youn, Tae-Seok;Shin, Hi-Jung;Noh, Gyeong-Woon
    • The Korean Journal of Nuclear Medicine Technology
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    • v.21 no.2
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    • pp.28-30
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    • 2017
  • Purpose Anti-Glutamic acid decarboxylase antibody test (GAD Ab) has been used as a predictor of type 1 diabetes. GAD Ab has also been shown to be highly potent in cerebrospinal fluid (CSF) of patients with suspected diabetic peripheral neuropathy. Recently, it has been known that clinical significance of GAD Ab using CSF is useful for the neurological disorders. However, the reference value of anti-GAD Ab has been provided only for serum. In this experiment, we estimated the reference value of anti-GAD antibody for CSF in neurological patients. Materials and Methods A total of 211 neurological patients were enrolled. Serum and CSF were analyzed by radioimmunoassay (RIA) using commercial RIA anti-GAD Ab kit (RSR, London, United Kingdom). Normal saline was used as the normal CSF control because CSF is most similar to 0.9% normal saline. Results The mean value of normal CSF control was 1.97 U/mL, and two standard deviations (2SD) was 1.44 U/mL. Based on this data, the expected reference range of CSF could be estimated from 0.54 U/mL to 3.40 U/mL Conclusion The reference range of normal CSF control using normal saline obtained with Hoffmann's method. However, there will be a need to validate the CSF reference values using human normal CSF.

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Adenovirus-mediated mGM-CSF in vivo Gene Transfer Inhibits Tumor Growth in a Murine Meth A Fibrosarcoma Model

  • Kim, Sang-Hyeon;Suh, Kwang-Sun;Seong, Young-Rim;Choi, See-Young;Rho, Jae-Rang;Yoo, Jin-Sang;Hwang, Kyeng-Sun;Cho, Won-Kyung;Im, Dong-Soo
    • The Journal of Korean Society of Virology
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    • v.30 no.2
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    • pp.141-150
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    • 2000
  • The effectiveness of noninfectious recombinant adenovirus encoding murine granulocyte-macrophage colony stimulating factor (mGM-CSF) for the treatment of Meth A fibrosarcoma was investigated in syngeneic BALB/C model. Meth A and HeLa cells transduced with the recombinant adenovirus (Ad.mGM-CSF) produced substantial amounts of mGM-CSF, while WEH1164 cells transduced with the virus did not produce mGM-CSF. Mice inoculated subcutaneously with $1{\times}10^6$ Meth A cells, followed by injection of Ad.dE1 as a control, developed large tumors that reached a mean tumor size of 22 mm by day 30. However, tumor development and tumorigenicity were significantly inhibited in mice with a single intratumoral injection of Ad.mGM-CSF at $1{\times}10^8\;pfu$. Histological examination of the tumors injected with Ad.mGM-CSF revealed dense infiltrates of neutrophils, histiocytes, lymphocytes, and eosinophils associated with apoptotic cell death. The results suggest that the recombinant adenovirus encoding GM-CSF have a potential use for cancer gene therapy.

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