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Objective To investigate whether toll like receptor ( TLR) signaling pathways can in-crease the expression of IL-17 R in neuralglial cells , and if they can whether the increased IL-17 R is func-tional.Methods Experimental autoimmune encephalomyelitis (EAE) was induced in B6 mice by immuni-zation with an emulsion of myelin oligodendrocyte glycoprotein 35-55 ( MOG35-55 ) in complete Freund's adju-vant (CFA).The expression of Il17ra and Il17rc in the brains and spinal cords of mice with EAE were de-tected by real-time PCR.Luxol fast blue ( LFB) staining was performed to the spinal cord sections to detect tissue demyelination.Immunohistological staining against IL-17RA and CD3 were undertook to visualize IL-17RA+and CD3 + cells.Same approaches were also applied to immunized Rag1 -/- mice to figure out whether T cells infiltration is necessary for increasing IL-17RA expression in the central nervous system ( CNS) .Then B6 mice were immunized with incomplete Freund′s adjuvant ( IFA) plus different TLRs ago-nists to measure the expression of Il17ra in the brains and spinal cords by qPCR .The purified astrocytes , microglia and oligodendrocytes isolated from neonatal mice brains were cultured in vitro for two weeks , and then treated with different TLRs agonists .The expression of Il17ra at mRNA and protein levels in the cells were determined by qPCR and Western blot respectively .The astrocytes were treated with IL-17A and LPS individually or in the combination to detect the level of CCL 2, CXCL8 and IP-10 in the supernatant by ELISA.Results B6 mice with induced EAE showed significantly increased Il17ra expressions in the brain and spinal cord , which was also detected in immunized Rag1 -/-mice.Although no spinal cord demyeliza-tion and CD3 cells infiltration were detected in Rag1 -/-mice, significantly increased number of IL-17RA positive cells could still be visualized .In vivo TLRs agonist participated immunization and in vitro treatment of purified neuroglial cells demonstrated that TLRs agonists could directly evoke IL -17RA expression in the CNS or cultured astrocytes , microglia and oligodendrocytes with high efficiency .Both IL-17 A and LPS could stimulate astrocytes to secrete CCL2, CXCL8 and IP-10, however, a combined use of IL-17A and LPS fur-ther augmented the production of these chemokines to a large extend .Conclusion Taken together , we con-cluded that TLRs agonists could directly stimulate neuroglial cells to express IL -17RA which functionally re-spond to IL-17A by secreting chemokines .
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Objective To study whether osteoblast is necessary for IGF-Ⅰ to promote bone resorption by osteoclast.Methods Mouse MC3T3 osteoblast cells and mature osteoclasts induced by RANKL were cultured in vitro.These osteoblasts and osteoclasts were subjected to treatment with recombinant human insulin-like growth factor-1 (rhIGF-Ⅰ),and the activation of IGF-Ⅰ receptor was verified by Western blotting.Thereafter,osteoclasts were cultured individually or co-cultured with osteoblast,in the absence or presence of rhIGF-Ⅰ.Osteoclast proliferation and apoptosis were observed by MTT colorimetric assay and flow cytometry.Cathepsin K gene expression was detected by real-time PCR; bone adsorption activity of osteoclast was determined by resorption pits formation on calf cortex slice with toluidine blue staining.Results Western blotting result confirmed that rhIGF-Ⅰ could effectively activate IGF-Ⅰ receptors either in osteoblast or osteoclast.In co-cultured group,in the presence of rhIGF-Ⅰ osteoclast showed inhibited apoptosis,enhanced proliferation and up-regulated cathepsin K expression (P ＜ 0.05).The functional experiment revealed that osteoclasts collected from IGF-Ⅰ treated co-cultured group resulted in more resorption pits formation (P ＜ 0.05); rhIGF-Ⅰ did not show any significant effect on the individually cultured osteoclasts.Conclusion Osteoblast is necessary for osteoclast induced bone resorption resulting from IGF-Ⅰ treatment.
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Objective To elucidate the expression diversity of IL-2R subunits in T helper(Th) cells under different activation condition and clarify its relationship with the response to IL-2 induced proliferation.Methods In vitro cultured Th1 cells were activated by plate bound anti-mouse CD3 McAb.In both activated and inactivated Th1 cells the expression of different IL-2R subunit was evaluated by real-time PCR and fluorescence staining,3H incorporation assay was adopt to measure the proliferation of the cells in response to IL-2 treatment,IL-2 affinity was determined via I125 labeled IL-2.IL-2Rα siRNA transfection was applied to knockdown CD25 expression in activated Th1 cells and its effect was confirmed by Western blot,IL-2 induced proliferation in IL-2Rα siRNA transfected Th1 cells were subsequently detected.CD4 cells were isolated from na(i)ve BALB/c mice and were also stimulated by plate bound anti-CD3 McAb.Cells were harvested at different days posterior to the stimulation,CD25 content and IL-2 induced proliferation were determined by flow cytometry and 3H incorporation assay respectively.Results In comparison with inactivated cells,only the expression of CD25but not other IL-2R subunits was significantly increased in activated Th1 cells.In accordance with elevated CD25,IL-2 affinity was also increased in activated Th1 cells,however,which resulted in decreased cell proliferation in response to IL-2 treatment.In activated Th1 cells,when CD25 expression was differently knockdown by different-dosed IL-2Rα siRNA transfection,the highest IL-2 response was observed in the cells with partially CD25 depression.Forin vitro activated na(i)ve CD4 cells the elevated CD25 expression gradually decreased and the highest proliferation response was detected at day 8 post stimulation upon IL-2 treatment.Conclusion Although CD25 is necessary for IL-2 induced proliferation,however,the excessively expressed CD25 lead to lower proliferation response.Not fully activated Thl or CD4 cells,but partially activated cells with lightly increased CD25 content possessed highest proliferation rate in response to IL-2 treatment.
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Objective To clarify whether the regulatory effect of all-trans-retinoic acid (atRA) on the antigen presentation function of dendritic cell(DC) is tightly associated with NF-κB signaling pathway.Also to clarify atRA mainly affected differentiated DC or influence the differentiation procedure from bone marrow cell to DC.Methods MOG35-55 immunized C57BL/6J mice received administration of atRA or not,splenic DC and CD4 cells isolated from immunized mice were subjected to in vitro cross-culture,treated with IL-12 and IL-23 respectively.Th1 and Th17 polarization of stimulated CD4 cells were determined by intracellular staining and FACS analysis,while the production of their corresponding cytokines,IFN-γ and IL-17,were measured by ELISA.Bone marrow cells were isolated from the femurs of the na(i)ve mice,treated with IL-4 and GM-CSF with or without synergistic RA treatment.MHC Ⅱ and CD11c molecules on the DC were assayed by immune staining and FACS analysis,their antigen presentation functions were decided by the proliferation and cytokine production of the Th1 effecter cells stimulated by antigen pulsed DC.To investigate the status of the NF-κB signaling pathway,the amount of phospho-Ser536 NF-κB p65 in the whole DC lysate and total NF-κB p65 in the nucleus were detected by Western blot.Finally,selective RA receptor antagonists were studied to figure out which receptor was majorly involved in the regulatory effect of RA on DC.Results Splenic DC from RA treated mice showed significantly decreased function of presenting antigen to stimulate CD4 cell polarization and cytokine production.Compared with untreated control,RA in vitro treated DC showed decreased expression of MHC Ⅱ and CD11c on its cell surface,which was accompanied with depressed function of stimulating Th1 cell proliferation and IFN-γ production.Further study revealed that RA mainly affect the differentiation procedure from BMC to DC,however,has no significant effect on differentiated DC as the aspects of MHC Ⅱ,CD11c expression,responder cell proliferation and cytokine production were evaluated.Decreased amount of phosphor-Ser536 NF-κB p65 in the whole cell lysate and total NF-κB p65 in the nucleus was investigated in RA treated DC,and decreased antigen presentation function of RA treated DC always came together with diminished activation of NF-κB signaling pathway.Finally it was demonstrated that RARβγ antagonist,but not RARα antagonist,could entirely block the RA effect on DC.Conclusion Retinoic acid inhibit the differentiation of DC as well as decrease its antigen presentation function,which might be resulted from the inactivation of NF-κB p65 signaling pathway and mediated by RARβγ.
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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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The mRNA expressions of chemerin and its receptor CMKLR1 in adipose tissue of OLETF rats and LETO rats were detected by real-time PCR. The results showed that the two genes mRNA expressions in subcutaneous and visceral adipose tissue in OLETF group were significantly higher than those in LETO group (P< 0.05 or P<0.01) and expressions of them in visceral adipose tissue were higher than those in subcutaneous adipose tissue(P<0. 05 or P<0. 01), suggesting that chemerin and CMKLR1 may be involved in the pathogenesis of obesity, insulin resistance, and type 2 diabetes mellitus.
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Objective:To identify the immune characteristics elicited by immunogen prepared in two different ways against a 36AA-peptide. Methods：Rabbits were immunized by a 36-AA-hapten coupled with carrier proteins or recombinant fusion protein separately. The kinetics and specificities of antibody responses were compared. Results：After about 3 months of primary immunity, the antisera elicited by two kinds of immunogen reached the peak titer. The highest titer elicited by the immunogen prepared with coupled-hapten method reached 1∶12 000, while the highest titer elicited by the immunogen prepared with gene engineering method was 1∶6 000. But the immune response produced by the recombinant fusion protein was more long-lasting. Conclusion：Both kinds of immunogen can successfully elicit 36-AA specific antibody response in rabbits, which have different immune characteristics. To couple the hapten with vector proteins is a quicker and more effective way to prepare the immunogen.
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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.
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Objective To discuss the value of medium resolution typing method for HLA-B antigens of Northern Chinese by DNA chip technique.Methods The chip was made with specific medium distinguish typing probes designed according to gene frequency of HLA-B alleles from Northern Chinese.Unsymmetrical PCR was used to amplify HLA-B exon 2 and 3, then labeled PCR products and hybridize with probes on the chip.Certified the typing of HLA-B by analysed and scanned the signals of hybridize through a set of computer software.Results HLA-B alleles were successfully typed in 30 clinical samples. This medium distinguish probes were able to discern 42 HLA-B alleles from the scope of HLA-B 7~83. Using it we can distinguish B14,73 and 82 three new alleles contrast to PCR-SSP methods.Conclusions DNA typing of HLA-B by chip was proven to be a high-resolution and high-specificity method. It is able to check out the multitudinous samples in one DNA chip and it is more suitable for clinical application.
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Objective:To establish a DNA typing method for HLA-I antigens with medium resolution method by DNA chip technique.Methods:The chip was made with specific medium distinguish-typing probes designed according to gene frequency of HLA-I alleles from Northern Chinese. Unsymmetrical PCR was used to amplify HLA-I exon2,3,and then the PCR products labeled and hybridized with probes on the chip.Typing of HLA-I was certified by scanning the hybridizing signals of through a set of computer software.Results:HLA-I alleles were successfully typed in 30 clinical samples .This medium-distinguishing probes were able to discern 57 HLA-I alleles accurately.Conclusion:DNA typing of HLA-I by chip has been proven to be a high-resolution and high-specific method. It is able to check out the new alleles that can not be distinguished by other methods with the same resolution., and it is more intuitional and more suitable for clinical application .