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Objective To explore the role and mechanism of inducible nitric oxide synthase inhibitor 1 400W in alleviating ischemia-reperfusion injury of human intrahepatic bile duct epithelial cells.Methods Human intrahepatic bile duct epithelial cells (HIBEC) in logarithmic phase were inoculated into culture plate at an appropriate density.The samples were randomly divided into control group (group C),ischemiareperfusion group (group I/R) and ischemia-reperfusion + 1 400W group (group I/R + 1 400W).Group C was cultured routinely;cells in I/R and I/R + 1 400W groups were placed in a three-gas incubator for 12h for simulating ischemia and then normal culture for 6h for simulating reperfusion.The I/R + 1 400W group had a final concentration of 100 μmol/L of 1 400W before ischemia and hypoxia.After reperfusion,cells and culture medium were collected,CCK 8 was used for detecting cell vitality,microplate method for detecting the content of lactate dehydrogenase (LDH) in culture medium,AnnexinV-FITC/PI double stain for detecting apoptosis level,Western blot for analyzing the expressions of endoplasmic reticulum stress (ERS)related protein cysteinyl aspartic acid protease 12 (caspase-12),glucose regulatory protein 78 (GRP78) C/EBP homologous protein (CHOP) and inducible nitric oxide synthase (iNOS).Results As compared with group C,cell viability significantly decreased in I/R and I/R+ 1 400W groups (53.8％ ± 2.3％ vs.100％,66.5 ％ ± 2.8 ％ vs.100 ％) (P＜0.05) while LDH increased markedly in cell culture medium (287.4 ±9.0U/L vs 120.2 ± 8.7U/L,212.0 ± 8.3U/L vs 120.2 ± 8.7U/L) (P＜0.05).Apoptosis accelerated markedly (41.5％±2.3％ vs5.2％±0.5％,32.7％± 1.8％ vs 5.2％±0.5％) (P＜0.05) and the expressions of caspase-12,GRP78,CHOP and iNOS spiked (P＜0.05);as compared with I/R group,cell viability of I/R+ 1 400W group rose while LDH,apoptosis level,caspase-12,GRP78 and CHOP declined in cell culture medium (P＜0.05).Conclusions 1 400W may alleviate ischemia-reperfusion injury of human intrahepatic bile duct epithelial cells and its mechanism may be correlated with a suppression of endoplasrnic reticulum stress.
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Objective:To analyse the biological function of anti-IL-6Rβ(gp130) monoclonal antibody and its regulatory effect on IL-6 signaling.Methods:Biological characteristics of anti-IL-6Rβ(gp130) mAb were assessed by Western blot analysis, capture ELISA and peptide ELISA .The phosphorylation of STAT 3 was tested by Western blot analysis in IL-6-stimulated U266/RA-FLS/RA-PBMC with or without anti-IL-6Rβ(gp130) mAb treatment.Results:3 strains of mouse anti-human gp130 mAb were with high affini-ty and different binding epitopes , the kaff of 10A1 was 2.62E-10.In U266, RA-PBMC and RA-SFMC, IL-6 signaling highly activated STAT3 which could be inhibited by anti-gp130 mAb.Conclusion: Anti-IL-6Rβ( gp130 ) mAb might have different binding epitopes and could affect IL-6 stimulated phosphorylation of STAT3, which provides a preliminary experiment for analyse the correlation of IL-6 signaling and RA .
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Objective: To investigate the variation of γδ T cells from healthy human peripheral blood(PB)and neonatus cord blood (CB)in proliferation and subtypes with isopentenyl pyrophosphate(IPP), and to acquire enough γδ T cells possessing distinct characteristics for experimental study.Methods: Mononuclear-cells from peripheral blood and cord blood induced by IPP were stained separately with monoclonal antibodies,which were fluorescein-labeled,and then used for assaying the expressing condition of surfaco molecules,as well as to evaluate the variation of γδ T cells on the percentage, subtypes and pbenotypes by FCM.Results:γδ T cells only account for a small proportion in both PB and CB.However,there was a significant difference in the heterogeneity between peripheral blood and cord blood γδ T cells.Vγ9Vδ2 T cells were dominant in peripheral blood γδ T cells.Most of Vγ9Vδ2 T cells in fresh isolated PBMC were central memory-type(CD27~+ CD45RA~-)and effector memory-type(CD27~-CD45RA~-)with IPP, PB γδ T cells proliferated strongly;The effector memory-typo(CD27~-CD45RA~-)turned into the main subtype in all Vγ9Vδ2 T cells,and HLA-DR and B7 molecules were highly expressed on the populations.But the cord blood γδ T cells showed rather complex subgroup heterogeneity,and Vγ9Vδ2 T cells were almost na(i)ve-type(CD27~+ CD45RA~+); Though γδ T cells were expanded(the percent of γδ T cells was increased),and Vγ9Vδ2 T cells were differentiated towards central memory-type and effector memory-type on day 14 with IPP,most of γδ T celLs still remained in the phase of na(i)ve-type(CD27~+ CD45RA~+).Conclusion:Tbere lies great differences of γδ T cells in quantity and subtypes between healthy person peripheral blood and neonatus cord blood.Peripheral blood γδ T cells can be activated and proliferated with IPP, while cord blood γδ T cells have the potential to deferentiate into director memory-type which can be used for experimental and clinical study with the synergy of corresponding cytokines.The immuno-regulation and effector function will be reported in other papers.
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Objective To establish the type Ⅱ collagen specific T cell line of Wistar rat and observe its effect on transferring arthritis.Methods The Wistar rats were immunized with emulsified chicken type Ⅱ collagen (CCⅡ) in complete Freund′s adjuvant by intradermal injection to induce the rat model of collagen induced arthritis (CIA).The lymphocytes were obtained from mesenteric lymph nodes of CIA rats,and the type Ⅱ collagen reactive T cell line was selected and propagated by CCⅡ stimulating in vitro .The proliferation response and phenotype were analyzed by 3 H TdR incorporation and fluorescence activated cell sorter (FACS).The onset of arthritis and pathological characteristic in ankle joints of recipient rats were observed with naked eye and histochemical examination.Anti CCⅡ antibody in serum was assayed by enzyme linked immunosorbent assay (ELISA).Results A T cell line was successfully established.The results of FACS labeled with fluorescent antibodies showed that 98 2% of the line cells were T cells,of which 89 7% were CD4 + T cells.The results of adoptive transfer showed that the incidence of arthritis was 50% when the injected cell number was 5?10 7,meanwhile the level of anti CCⅡ antibody in serum was elevated more than that of the control.Conclusion A cell line has been successfully established.The result of arthritis transferring by T cell line shows that the T cell plays a great role in the pathogenesis of CIA and provides a research datum for rheumatoid arthritis therapy with T cell vaccine.
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Objective To study regulatory mechanism of osteopontin (OPN) in rheumatoid arthritis (RA). Methods The expression of OPN in peripheral blood mononuclear cells (PBMC), synovial fluid cells (SFMC) and synovium tissue (ST) and T cell subsets from RA patients were detected by real time PCR. The concentration and relative rate of inflammatory factors in the synovial fluid from RA patients were analyzed by ELISA. Results The mRNA expression of OPN in synovial fluid and tissue was higher than that of PBMC in the same RA patient. The OPN expression was found mainly on CD4+T. The OPN concentration was higher in the synovial fluid than that of in the same patient′s serum. Meanwhile, the concentration of IL-10, IFN-? and TNF-? was higher than that of in the serum from same patient. Also, the concentration of IL-18 and IL-12 were higher than that of normal individual serum. Conclusion OPN may control secretion of inflammatory factors of synovium tissue and synovial fluid and induce the inflammatory response.
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Objective To assess the level of soluble Fas (sFas) and soluble Fas ligand (sFasL) in the sera of patients with systemic lupus erythematosus (SLE),rheumatoid arthritis (RA),multiple sclerosis (MS)and insulin dependent diabetes mellitus (IDDM).Method The level of sFas and sFasL was determined by a sandwich enzyme linked immunosorbent assay.Anti ssDNA (single stranded DNA) was detected by indirect enzyme linked immunosorbent assay.Results The level of sFas in patients with SLE,RA,IDDM and MS was significantly higher than that in healthy individuals.It was interesting that among these autoimmune diseases,the level of sFas in the sera of patients with SLE was dramatically higher than that of the other diseases.The high level of sFasL accompanied by sFas was found in the sera of SLE and RA patients.In the sera of patients with SLE,the anti ssDNA antibody always accompanied by high concentration of sFas and,by contrast,no anti ssDNA antibody was found in all the patients in whose sera no sFas was found.In patients with IDDM,the sFasL level of the serum was significantly lower than that of the serum of healthy donors.Conclusion Serum sFas level of patients with SLE,RA,MS and IDDM is higher than that of healthy individuals.These results indicate that the sFas level can be used as a marker of disease progress and relaxing after treatment with the medicine.It is also demonstrated that there is relationship between the level of anti ssDNA antibody and sFas.The level and significance of serum sFas and sFasL in these autoimmune disease patients are under investigation.