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Objective:To explore the immune infiltration cells in rheumatoid arthritis (RA) synovial lesions, and to provide new research directions and therapeutic targets for the pathogenesis and treatment of RA.Methods:The three gene expression data sets GSE77298, GSE55457 and GSE1919 were downloaded from gene expression omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo), and the data were merged with Perl. The "limma" package was used to adjust batch differences. In R, "CIBERSORT" software was used to obtain the expression matrix of 22 kinds of immune cells corresponding to RA synovial tissue samples and normal synovial tissue samples were analyzed with the three packages of "e1071", "parallel" and "preprocessCore". Perl was used to screen samples with P<0.05 in the immune cell matrix. R's "barplot" function was analyzed by the percentage of 22 immune cells in samples with P<0.05. The "pheatmap" package of R was used to visualize heatmaps, and "corrplot" package was used to draw correlation heatmaps. The "vioplot" package of R was used to draw violin plots of differences via the wilcox test. Results:The results of immune cell infiltration analysis showed that in RA synovial tissue samples and normal synovial tissue samples at P<0.05, B cells naive and natural killer cells resting were under-expressed in RA synovial tissue, and plasma cells, mast cells resting, macrophages M1, B cells memory and T cells regulatory were highly expressed in RA synovial tissue. This study also found that in the same sample, the correlation coefficient between natural killer cells resting and neutrophils ( r=0.91) was the highest, indicating synergistic effect between the two. In the same sample, the correlation coefficient between macrophages M0 and plasma cells ( r=-0.88) was the lowest, indicating antagonistic effect between the two. Conclusion:The immune infiltrating cells in RA synovial lesions discovered in this study provide a certain theoretical basis and research direction for the research on the disease mechanism and treatment of RA.
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Objective:To explore the potential Hub genes, key miRNAs, biological processes and related signaling pathways in the pathogenesis of osteoarthritis (OA), and provide bioinformatics basis for the pathogenesis and treatment of OA.Methods:The expression profiling chip of OA synovial tissue sample from Gene Expression Omnibus (GEO) were downloaded, differentially expressed genes (DEGs) were identified, and functional enrichment analysis was performed. A protein-protein interaction network (PPI) was constructed. STRING and Cytoscape was used for module analysis, and the Hub gene was further identified, and further miRNAs mining of the Hub gene was carried out.Results:Finally, 9 Hub genes (SOCS3, BTRC, FBXO32, KLHL22, UBE3A, HUWE1, UBR4, ANAPC5, TRIM50) and 2 key miRNAs (hsa-miR-103a-3P, hsa-miR-107) related to the progression of OA were identified .They might be potential biomarkers for the pathogenesis of OA. We also found that signal transduction, the transcriptional positive regulation of RNA polymerase Ⅱ promoter, and protein serine/threoninase activity had a certain correlation with the pathogenesis of OA. In addition, our analysis results showed that cAMP signaling pathway and Rap1 signaling pathway were also involved in the progression of OA.Conclusion:The potential biological molecules, biological processes and related pathways identified in this study may guide us for the further research on the etiology and treatment of OA.
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Objective To clarify the mechanisms that the response of fibroblast-like synovial (FLS) cellsto methotrexate (MTX) in rheumatoid arthritis (RA) and to provide theory basis for the drug treatment of RA.Methods Synovial fibroblasts were isolated from synovial tissue specimens obtained from patients with RA andexposed to MTX.Cell viability was measured using a MTT assay and cell apoptosis was valued by flow cytometry.Western blotting analysis of LC3 and immunocytochemistry were used to analyze the induction of autophagy in RA-FLS after treating with MTX.Transfection of siRNA was used to interfere the expression of Beclin1 to down-regulate the autophagy,cell apoptosis was valued by flow cytometry and western blot analysis was used to test the PARPp85 with or without the presence of MTX.Statistical product and service solutions (SPSS) 18.0 statistical software was used for statistical analysis of all experimental data.Independent sample t test was used according to data distribution status,homogeneity of variance,and normal distribution.GraphPad Prism 5.0 was used to draw statistical graphs.Results MTX induced apoptosis was increased in RA-FLS.MTX stimulated the autophagy response in RA-FLS by inducing autophagosome formation.In RA-FLS,transfection with Beclin1 siRNA inhibited autophagy and increased the susceptibility to MTX,which induced cell death.Conclusion Autophagy of RA-FLS contributes to the resistance to apoptosis induced by methotrexate.
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Objective To clone and fuse the cDNA of human cytokeratin 9 in prokaryotic expression system,and purify and identify the fusion protein.Methods The cDNA fragment of human cytokeratin 9 was amplified from human keratinocyte (HaCaT) total RNA with specific primers.The PCR products were cloned into vector pET-28a,then the fusion protein of his-CK9 was induced by IPTG.The expressed fusion protein of his-CK9 was purified by nickel ion affinity chromatography and identified by SDS-PAGE and Western blot.Results The sequencing proved that the recombinant vector of the cDNA of CK9 was correct.The fusion protein of his-CK9 was induced to be expressed in E.coli.The fusion protein of his-CK9 was highly purified and his-CK9 showed specific binding to the commercialized antibodies of CK9.Conclusion The recombinant vector of pET-28a-CK9 has been successfully constructed,and the fusion protein of his-CK9 has been successfully expressed and purified.
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Objective To investigate the etiologic roles of apoptosis-associated genes,environmental factors and their interactions in lumbar disc herniation (LDH).Methods A case-control trial was conducted.We recruited 128 outpatients with LDH as case group and 132 normal people matched by age and gender as control group.Peripheral venous blood samples were collected and DNA was extracted from leukocytes.By using a modified Brucker Autoflex MALDI-TOF mass spectrometer,we analyzed 3 genes with 9 polymorphic sites,namely,Fas-1377G/A rs2234767,Fas-670G/A rs1800682,Fas rs2147420,Fas rs2296603,Fas rs7901 656,Fas rs1 57101 9,FasL-844C/T rs7631 10,CASP-9-1263A > G rs4645978,and CASP-9-712C > T rs4645981.The correlations between polymorphism of Fas,FasL and CASP-9 genes and the risk of LDH were evaluated by non-conditional Logistic regression model.Multiple Logistic regression model was performed to assess the interaction between apoptosis-associated genes and environment factors,such as lumbar vertebral loads,bed type,spare-time exercises and spare-time activities. Results There were preferable balances in case and control groups in age and gender without significant differences.However,the two groups differed significantly (P G (rs4645978),and FasL-844C/T TT and CASP-9-1263A>G GG genotypes might be the high risk genotypes of LDH.The gene-environment interaction analysis revealed that super-multiplicative and sub-multiplicative interactions respectively between FasL-844TT genotype and lumbar vertebral loads (3-4 level),and between CASP-9-rs4645978 GG and lumbar vertebral loads (3-4 level).Conclusion FasL,CASP-9 genes and lumbar vertebral loads and their interactions play important roles in the pathogenesis of LDH.It suggests that the risk of LDH may be codetermined by environmental factors and inherited susceptibility genes,and that the mechanisms of interactions vary in different genotypes and the same or different environmental factors.