Triptolide inhibites Th17 cell differentiation via regulating cyclooxygenase-2/ prostaglandin E2 axis in synovial fibroblasts from rheumatoid arthritis / 中国中药杂志

Triptolide (TPT), an active compound extracted from Chinese herb Tripterygium wilfordii , has been used in therapy of rheumatoid arthritis (RA). In this study, after synovial fibroblasts from rheumatoid arthritis (RASFs) were treated with TPT, we investigated its effect on the differentiation of Th17 cells. Firstly, the mRNA level of cyclooxygenase (COX) wad detected by qRT-PCR and the protein level of prostaglandin E2 (PGE2) was tested by ELISA in RASFs treated with different concentrations (0, 10, 50, 100 nmol L-1 ) of TPT. Then after TPT pre-treated RASFs and RA CD4 + T cells wer e co-cultured for 3 days in the presence or absence of PGE2, IL-17 and IFN-gamma production in CD4 T cell subsets were detected by flow cytometry. The results showed TPT decreased the mRNA experssion of COX2 and the secretion of PGE2 in RASFs in a dose-dependent manner(P <0. 05). We further found that differentiation of Thl7 cells was downregulated in a dose-dependent manner, and exogenous PGE2 could reverse the inhibition of Th17 cell differentiation(P <0. 05). Taken together, our results demonstrated that TPT inhibited the mRNA level of COX2 and the secretion of PGE2 in RASFs, which partly led to impaired Th17 cell differentiation in vitro.

Expression of recombinant extracellular region of human interleukin-1 receptor type I in Pichia pastoris / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct pPICZalphaA-soluble interleukin-1 receptor type I (sIL-1RI) recombinant expression vector containing the gene fragment encoding the extracellular domain of sIL-1RI for its expression in Pichia pastoris.</p><p><b>METHODS</b>sIL-1RI gene was amplified by RT-PCR and inserted into the yeast expression vector pPICZalphaA by digestion ligation. The recombinant plasmid pPICZalphaA-sIL1RI was transformed into E.coli Stb13, and the positive clones were analyzed by PCR and DNA sequencing. The pPICZalphaA-sIL1RI recombinant plasmid was electroporated into GS115 cells and the transformants were analyzed by PCR. After phenotype identification, the recombinant strains were induced by methanol to express the target protein, which was analyzed by Western blotting of the cell extract and supernatant.</p><p><b>RESULTS</b>The recombinant plasmid pPICZalphaA-sIL-1RI was constructed successfully, and the results of Western blotting showed that yeast induced by methanol expressed a protein of about 39 kD.</p><p><b>CONCLUSION</b>sIL-1RI protein has been successfully expressed in P.pastoris expression system, which provides the basis for further study of sIL-1RI.</p>

Isolation and characterization of human rheumatoid arthritis fibroblast-like synoviocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To isolate and characterize human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs).</p><p><b>METHODS</b>The synovial membrane tissues were obtained from 4 RA patients, 1 chondroma patient and 1 healthy subject and FLS were isolated by means of tissue culture. The cell morphology was observed by phase-contrast microscope and the cell surface markers were detected by flow cytometry.</p><p><b>RESULTS</b>The FLSs were successfully cultured from the synovial membrane tissues with good cell homogeneity after the third passage. The FLSs of the 3rd to 7th passages were stable and proliferated actively, followed by slow proliferation and aging since the 8th passage. Flow cytometry showed that the 4th-passage FLSs from the RA patients contained 99.04% CD90(+) cells, 2.73% CD3(+) cells, 0.29% CD3(-)CD19(+) cells, 2.81% CD3(-)CD16(+)CD56(+) cells, 5.89% CD14(+) cells, and 54.17% CD55(+) cells. The presence of interleukin-1 receptor type I (IL-1RI, 158.63-/+20.32 pg/ml) and IL-1beta (4.67-/+0.82 pg/ml) were detected in the cell culture supernatant of the 4th-passage FLSs from the RA patients by enzyme-linked immunosorbent assay ELISA.</p><p><b>CONCLUSION</b>FLSs from RA patients can be effectively culture by means of tissue culture, and the cultured FLSs show high expressions of CD90, IL-1RI and IL-1beta.</p>

Assessment of the performance of an automated analysis system in detecting C3 and C4 / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the performance of BNII auto-analyzer system in detecting C3 and C4.</p><p><b>METHODS</b>CLSI protocols (EP15-A, EP6-A, EP9-A2) and other relevant literatures were use to or evaluate the precision, accuracy, linearity of C3 and C4 detection by the auto-analyzer system, and the results were compared with the recognized standards.</p><p><b>RESULTS</b>The relative bias of C3 and C4 was less than one third of the CLIA'88 standard and the precision met the clinical requirement. The results tested by DADE BNII system were not compatible with those by Roche Modular System. C3 showed good linearity in the tests (R2>0.975, P<0.05) with a linearity range of 0.18-5.1 g/L. The linearity of C4 was not available because of lack of high-level samples.</p><p><b>CONCLUSION</b>The performances of DADE BNII System basically meet the recognized standards in clinical detection of C3 and C4, but the method comparison needs further validation.</p>

Determination of IgM-RF, IgA-RF, IgG-RF, IL-1RI, CDK2 and ROC evaluation in patients with rheumatoid arthritis / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the value of rheumatoid factors IgM-RF, IgA-RF, IgG-RF, interleukin-1 receptor type I (IL-1RI) and cyclin-dependent kinase 2 (CDK2) in the diagnosis of rheumatoid arthritis (RA).</p><p><b>METHODS</b>IgM-RF, IgA-RF, IgG-RF, IL-1RI and CDK2 were detected in 47 patients with active RA, 47 with inactive RA, 20 with active systemic lupus erythematosus (SLE), 20 with inactive SLE, 20 with acute upper respiratory tract infection (AURTI), and 20 healthy controls using enzyme-linked immunosorbent assay (IgM-RF, IgA-RF, IgG-RF, IL-1RI) and time-resolved fluoroimmunoassay (CDK2).</p><p><b>RESULTS</b>Patients with active and inactive RA showed significant differences in peripheral serum CDK2 and IgA-RF levels (P<0.05). Between active and inactive RA patients, RA patients and SLE patients, RA patients and AURTI patients, and RA patients and the control subjects, the area under curve (AUC) of the receiver operating characteristic curve (ROC) was 0.561, 0.814, 0.799, and 0.888 for IgM-RF, 0.596, 0.678, 0.729, and 0.850 for IgA-RF, 0.614, 0.718, 0.692, and 0.791 for IgG-RF, 0.646, 0.691, 0.762, and 0.835 for IL-1RI, 0.803, 0.753, 0.741, and 0.840 for CDK2, respectively.</p><p><b>CONCLUSIONS</b>IgM-RF may have high value in the diagnosis and differential diagnosis of RA, and CDK2 can be useful for differential diagnosis between active and inactive RA.</p>

Association of interleukin-6 and interleukin-18 gene polymorphism with rheumatoid arthritis in Guangdong Han population / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the single-nucleotide polymorphisms (SNPs) at positions -572 and -174 in the promoter region of interleukin-6 (IL-6) gene and at -607 and -137 in the promoter region of interleukin-18 (IL-18) gene for their association with rheumatoid arthritis (RA) in the Chinese Han population in Guangdong Province.</p><p><b>METHODS</b>SNPs of IL-6 and IL-18 genes were detected in 120 patients with RA and 168 normal subjects using polymerase chain reaction with sequence-specific primers (PCR-SSP).</p><p><b>RESULTS</b>SNPs at -572 and -174 of IL-6 gene and at -607 and -137 of IL-18 gene were detected in this population. There was a significant difference in the genotype and allele frequency at -572 and -174 of IL-6 gene and -607 of IL-18 gene (P<0.001), but not in the distribution of genotype frequencies at -137 of IL-18 gene between the RA patients and healthy subjects (P=0.141). A significant difference was found, however, in the allele frequency at -137 of IL-18 (P=0.024). Logistic regression analysis revealed significant association of age, gender, IL-6 gene -572, -174 and IL-18 gene -137 SNPs with RA (P<0.05).</p><p><b>CONCLUSIONS</b>The polymorphisms of the promoter region of IL-6 gene at positions -572 and -174 is probably associated with RA, and further study is needed to understand the relation of the polymorphisms of IL-18 gene at positions -607C/A and-137G/C with RA.</p>

Method performance verification of the clinical chemiluminescence immunoassay / 中华检验医学杂志

Objective To establish a method performance verification project and experimental method for the clinical chemiluminescence immunoassay.Methods Referring to CLSI evaluation protocols and pertinent literature,and by combining our actual works,we designed a verification procedure and experimental method.By Using these above,the precision,accuracy,analytical sensitivity,analytical measurement range,clinical reportable range and biotic interval of AFP on the Bayer Centaur 240 chemiluminescence immunoassay system were verificated.Results would be compared with the declaration of the manufacturer or desirable specifications derived from biologic variation.Results The results showed that the between-day inaccuracy on AFP levels at 77.4 ng/ml and 168.0 ng/ml was 5.70% and 4.84% respectively,these were consistent with manufacturer's inaccuracy claimed.The relative bias between the results measured for calibrator at four levels and target value was less 5.0%,and the relative bias between the results measured for EQA control sample at five levels and target value was-3.4% to 11.9%.Lower limit of detection was 1.04 ng/ml,lower slightly manufacturer's analytical sensitivity claimed.Biologic limit of detection was 2.65 ng/ml-3.53 ng/ml,functional sensitivity was 3.53 ng/ml.Analytical measurement range was 3.53-912.00 ng/ml,within manufacturer's liner range claimed.Clinical reportable range was 3.53-182 400.00 ng/ml.Reference interval was 0.6-7.7 ng/ml,within manufacturer' s claimed.Conclusions The main performances of the detection system are accorded with the declaration of the manufacturer.The performance verification procedure and experimental method of our research ars simple and practical,which has important significations for building medical laboratory and laboratory accreditation, improving quality of the chemiluminescence immunoassay.