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Objective To investigate the clinical application value of fluorescence polymerase chain reaction (PCR) .in the human leucocyte antigen-B27(HLA-B27) gene and gene typing detection of ankylosing spondy-litis (AS) patients .Methods A total of 43 clinical blood samples of AS and 56 samples of healthy controls were collected in Shenzhen Futian hospital for rheumatic diseases from January 2014 to March 2015 .HLA-B27 gene was detected by flow cytometry .HLA-B27 gene and gene typing was also detected by the fluorescence PCR method .Results Among 43 samples ,40 samples were HLA-B27 positive(93 .02%) by flow cytometry while 39 samples were HLA-B27 positive (90 .70%) by fluorescence PCR .The total coincidence rate was 97 .50% .Among 39 positive samples ,32 samples were HLA-B2704 positive (82 .05%) and 7 samples were HLA-B2705 positive (17 .95%) .Conclusion The fluorescence PCR is an accurate method to detect HLA-B27 gene and presents high consistency with flow cytometry .It can also detect the HLA-B27 gene typing .It may have great clinical application value and prospects .
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Objective To investigate the expression of miRNA-16 in peripheral blood monouclear cells (PBMC)from systemic lupus erythematosus( SLE)patients. Methods Sixteen SLE patients who meet the diagnostic criteria of SLE revised in 1997 American rheumatology and 12 healthy individuals were selected as our subjects. Their peripheral blood were sampled. Total RNAs were extracted and purified. The level of miRNA-16 was determined by quantitative reverse transcription PCR( qRT-PCR). U6 was used as housekeeping control. The amount of target miRNA was normalized relative to the amount of U6(ΔCt =ΔCt miRNA-ΔCtU6 ). Relative expression levels were expressed as 2-ΔCt . Results The expression level of miRNA-16 in the SLE patients was 919. 87 ± 715. 45,significantly higher than that in the healthy control group(413. 6 3 ± 330. 69;t= -2. 497,P﹤0. 05). And miRNA-16 expression in SLE active group was 1 298. 79 ± 803. 79,significantly higher than that in SLE stable group(540. 95 ± 350. 15;t= -2. 445,P﹤0. 05). The level of miRNA-16 was related with AnuA (r=0. 669,P=0. 005),ESR(r=0. 608,P=0. 012)and SLEDAI(r=0. 530,P=0. 035). Conclusion The expression of miRNA-16 is high in SLE patients and it is related with SLE activity.
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Objective To elucidate the function way of micro RNA(miR)-155 in the differentiation of Th17 cells.Methods CD4+T cells were separated from mice spleens using MACS CD4+T cells separatinge kit and cultured with interleukins [interleukin (IL)-2, IL-23 and IL-6] which could induce CD4+ T cells differentiate into Th17 cells.IL-17 was detected by flow cytometry and enzyme linked immunosorbent assay (ELISA) after transfected with miR-155 mimics or inhibitor lentiviral vectors.The expression levels of miR-155, IL-17A mRNA and Ets-1 mRNA were detected using fluorescent quantitation real-time quantitative polymerase chain reaction (RT-PCR).The si-Ets and miR-155 co-function for Th17 differentiation was analyzed.Data analysis was perfoemed using one-way analysis of variance (ANOVA) test and Dunnett test for pair-wise comparison and t test.P＜0.05 was considered to be statistically significant.Results The CD4+T cells were divided into four groups (the untreated control untreat group, the treatment control treat group, the miR-155 mimnics group and miR-155 inhibitor group).IL-17 was scarcely expressed and secreted in the untreated control untreat group.The cells expression of IL-17 were significantly different among the four groups (F=160.549, P＜0.01).The cells expressing of IL-17 were higher in the miR-155 mimics group (39.86±4.62)％ than those at the miR-155 inhibitor group (22.02±2.81)％, P＜0.01) and in the treated control treat group [(19.44±1.49)％, P＜0.01].The level of IL-17 was also significantly different among the four groups (F=260.813, P＜0.01).The level of IL-17 was higher in the miR-155 mimics group [(1 509±136) pg/ml] than that in the miR-155 inhibitor group [(923± 42) pg/ml, P＜0.01);and in the treated control group [(767±94) pg/ml, P＜0.01).The expression of miR-155 (12.53±0.80 vs 1.78±0.14, 7.16±0.62, 6.47±0.92, P＜0.01) and IL-17A mRNA (46.55±6.71 vs 1.01±0.19,15.62±1.26, 14.20±2.73, P＜0.01) was significantly higher than that in the other three groups, while the expression of Ets-1 mRNA was significantly lower (0.66±0.10 vs 1.19±0.04, 1.01±0.16, 1.37±0.27, P＜0.01).si-Ets-2 was screened because it markedly inhibited the expression of Ets-1 mRNA among the three designed siRNAs.The expression of IL-17A mRNA was higher (17.19±3.58 vs 10.08±0.76, t=-3.361, P=0.028) and the expression of Ets-1 mRNA was lower (0.27±0.01 vs 0.74±0.03, t=-30.275, P＜0.01) in si-Ets-2 group than that in si-Con group when si-Ets-2 or si-Con was co-transfected with miR-155 mimics or inhibitor lentiviral vectors.The expression of Ets-1 protein was lower in si-Ets-2 group than that in si-Con group by Western blotting and the decrease was markedly obvious in the miR-155 mimics group.Conclusion miR-155 can induce CD4+T cells to differentiate into Th17 cells by inhibiting the gene expression of Ets-1.
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ObjectiveTo investigate the expression of miR-155 and miR-146a in peripheral blood mononuclear cells (PBMC) and plasma of rheumatoid arthritis (RA) patients.MethodsPBMC and plasma were separated from the peripheral blood of 34 RA patients and 15 healthy individuals.Total RNAs were isolated and miRNAs were purified.The levels of miR-155 and miR-146a were determined by quantitative reverse transcription PCR(qRT-PCR).U6 was used as housekeeping control.The amount of target miRNA was normalized relative to the amount of U6(ΔCt=ΔCtmiRNA-ΔCtU6).Relative expression levels were expressed as 2 △-ΔCt.Data were analyzed using SPSS 13.0 software.The test of homogeneity of variance and unpaired t-test was used to compare between groups.P values(2-tailed) less than 0.05 were considered as statistically significant.ResultsThe expressions of PBMC and plasma miR-155 were higher in RA patients than those in the healthy control individuals(0.08±0.08 vs 0.05±0.03,t=-2.225,P＜0.05; 5.9±6.7 vs 1.3±2.0,t=-3.677,P＜0.05).The expression of miR-146a in PBMC and plasma of RA patients and controls were(1.3±1.2 vs 0.8±0.6,t=-2.154,P＜0.05)and(741±1001 vs 300±295,t=-1.669,P＞0.05).According to their DAS28 value,RA patients were divided into high activity group (23 cases,DAS28≥5.0) and low disease activity group( 11cases,DAS28＜5.0).The plasma miR-155 and miR-146a expressions were significantly higher in high activity group than those in low activity group.There were no significant differences in the expression of PBMC miR-155 and miR-146a between the two groups.ConclusionThe expression of PBMC and plasma miR-155 and miR-146a are higher in RA patients.The expression of plasma miR-155 and miR-146a are associated with RA patients' activity.Plasma miR-155 and miR-146a may be potential non-invasive biomarkers for RA diagnosis anddisease activity assessment.
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In this paper, we designed to investigate the frequencies of tumor necrosis factor receptor 2 (TNFR2) polymorphisms at nt587 and nt694 in south Chinese SLE patients and healthy individuals and explore whether genetic variants in TNFR2 gene is involved in the pathogenesis of SLE. The results showed that the nt587G allele frequency was 21.1% in the 128 SLE patients and the allele frequency was 13.0% in the 135 healthy individuals, the former was significantly higher than the latter in the allele frequency (P ＜ 0.05). People with the nt587 G variant showed high risk to SLE. The frequency of nt694 was slightly but not statistically significantly increased in SLE patients compared with healthy controls(16.0% versus 11.9%, P= 0.149). These results indicate that the polymorphism at nt587 of TNFR2 is associated with the south Chinese SLE patients. The polymorphism at nt694 is not associated with SLE.