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Objective To investigate the expression of adenosine receptor (ADOR) subtypes (A2A and A2B subtypes) in the mucosal dendritic cells (DCs) from patients with Crohn's disease and their pathogenic roles.Methods Mucosal DCs (mDCs) were isolated from resected intestine of patients with or without Crohn's disease.Some of the mDCs were cultured in vitro and others were used to extract RNA.The expression of ador-a2a and ador-a2b were detected by real-time qPCR.mDCs in culture were treated with selective ADOR-A2A and ADOR-A2B agonists (CGS 21680 and BAY 60-6583) and then the concentration of IL-1,IL-6 and IL-12 in the medium were measured by ELISA.The binding affinities of ADOR-A2A and ADOR-A2B to adenosine were determined by 3H-adenosine in combination with selective ADOR-A2A and A2B antagonists (SCH58261 and MRS1706).Na(i)ve CD4+ cells were collected from human umbilical cord blood and co-cultured with mDCs treated by different ADOR agonists to observe T cell responses.The production of cytokines in culture was measured by ELISA.The polarization of CD4+ cells was analyzed by intracellular cytokine staining and FACs analysis.Peripheral blood mononuclear cells (PBMCs) were treated with IL-4 and GMCSF to induce the expression of monocyte-derived DCs (Mo-DCs).Mo-DCs were treated with different toll-like receptor ligands to investigate their effects on the expression of ador-a2a and adora2b.Moreover,Mo-DCs were treated with LPS and BAY 60-6583 individually or in the combination to stimulate CD4+ cells.Then the production of cytokines and the polarization of CD4+ cells were evaluated.Results Compared with patients without Crohn's disease,patients with Crohn's disease showed no change in the expression of ador-a2a but a significantly increased expression of ador-a2b in mCDs (CD-mDCs),enabling to bind more adenosines.Activated ADOR-A2B signaling pathway induced CD-mDCs to secret more proinflammatory cytokines and to promote polarization of CD4+ cells toward Th1 and Th17 cells.Toll-like receptor ligands,pam3csk4 and LPS could intensively augment the expression of ador-a2b in Mo-DCs.The pathogenicity of Mo-DCs was strengthened upon a combined stimulation with BAY 60-6583 and LPS.Conclusion The significantly increased expression of ador-a2b in mDCs might be involved in the pathogenesis of Crohn's disease by promoting mDCs to secret more pro-inflammatory cytokines and enhancing the polarization of CD4+ cells.Moreover,the expression of ador-a2b in DCs could be regulated by certain toll like receptors.
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Objective To examine the expression of thyroid hormone receptor isoforms (TR α1, α2, β1, and β2) in human osteoblast-like cell line MG-63 at the mRNA level and the effect of thyroid hormone (T3 or T4 ) on the expression. Methods Realtime quantitative PCR was performed. Results The expression of TRα1 mRNA was the highest, that was 10. 70± 0.45, TRβ1 was 5.75 ± 0. 10, TRβ2 was 3.34 ± 0. 08, and TRα2 was very low, only (3.66 ±0. 59) × 10-2. Only the expression of TRαl and TRα2 mRNA was down regulated significantly by the treatment of 10-10 ～ 10-6 mol/L T3, and there was a negative correlation between the expression of TRα1 or TRα2 mRNA and the concentration of T3. Conclusion TRα1 plays a primary role in mediating the effects of thyroid hormones in skeletal development.
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To establish a method for radioligand binding assay of thyroid hormone receptors(TR)in human osteoblast-like osteosarcoma cell line MG-63 and to estimate the kinetic parameters of putative receptors. The MG-63cell was cultured in Ham's F12, the soluble TR was prepared from the intact nuclear extracts. The binding properties between TR and T3 were performed by using the traditional Scatchard analysis. The apparent Ka of TR in MG-63 is 7.68× 109 L/mol, and MBC(111. 25+ 10.77)fmol/mg protein. The study indicated that MG-63 cells possessed high affinity and limited-capacity of TR in its nuclear extracts. This may serve as the starting and basic work about TR in bone cell.
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To study the transactivation of a novel thyroid hormone receptor isoform, TR?. pcDNA3. 1-TR? and pGL3-Promoter/thyroid hormone responsive element were co-transfected into COS-7 cells. The expression of reporter gene was detected. It could be increased up to 45 times by T3. TR? and showed the characteristics of a transcription factor.
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Objective:To construct suitable vectors for the secretory expression of hirudin in Pichia pastoris.Methods:The α-facor-hirudin gene was amplified from pPIC9-hirudin by PCR and sub-cloned into PA0815.The multi-copy recombinant plasmid pA0815-(α-Hirudin)n was constructed.The recombinant was transformed into P.pastoris strain GS115 for induction expression and then the activity of secreted products was identified.Results:A new multi-copy vector pA0815-(αHirudin)n was successfully constructed and was capable of secreting recombinant hirudin efficiently,which was confirmed respectively by PCR and SDS-PAGE.The products possessed the activity of thrombin inhibitor.Conclusion:This result offers efficient P.pastoris stains for mass production of biological active hirudin.
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Objective:To construct an E.coli expressing system of human interferon-?(IFN-?).Methods:Extracted DNA from human blood and PCR human IFN-?, cloned the human IFN-? gene into plasmid T-easy and pBV-220. Expressed human IFN-? in E.coli DH5?, the expressing product was analysed by SDS-PAGE, Western blot and anti-virus capacity test.Results:DNA sequence analysis showed the recombinant plasmid pBV- IFN-? contained human IFN-?. SDS-PAGE and Western blot proved that there were hIFN-? in E.coli DH5? after temperature inducing and the expressing product has anti-virus activity.Conclusion:A human IFN-? E.coli expressing system was constructed successfully, and the recombination human IFN-? has anti-virus activity.