| Part ? Study on gene expression profiles of TLR/MyD88 signaling pathway related genes in peripheral blood CD4 + T lymphocytes of patients with microscopic polyangiitis?Objective?In this study,we used gene chip technology to detect mRNA expression levels of TLR/MyD88-related genes in CD4+ T lymphocytes from peripheral blood of active stage patients with microscopic polyangiitis(MPA),and screened genes that were differentially expressed with healthy individuals.?Methods?A case-control study was conducted to collect 18 active stage patients with MPA(case group)and 16 healthy volunteers(control group)as subjects and collect the clinical data of the patients.CD4+ T lymphocytes were extracted from peripheral blood of all subjects.Total RNA was extracted from CD4+ T lymphocytes,and reversely transcribed in cDNA.Eight cases and six cases were randomly selected from the case group and the control group.A gene chip technology analysis was performed to study TLR/MyD88-related gene mRNA expression levels.The array test of partial differential genes wereverified by quantitative real-time qPCR(RT-PCR).SPSS 17.0 statistical software was used for statistical analysis.If the level of gene expression was up-regulated or down-regulated by more than 1.5 times compared with normal people,and P<0.05 was considered statistically significant.? Results ?(1)Compared with the healthy control group,19 of 84TLR/MyD88-related genes were differentially expressed in the active stage patients.Sixteen genes were up-regulated,up-regulated genes encompassed TLR4(3.46 times,P=0.0391)?TLR6(2.74 times,P=0.0064)?MyD88(1.57 times,P=0.0416)?TIRAP(1.77 times,P=0.0353)?TICAM1(1.73 times,P=0.0216)?TBK1(2.02 times,P=0.0064)?SOCS1(3.04 times,P=0.0022)?IL17A(4.66 times,P=0.0040)?RELA(1.78 times,P=0.0002)?NFRKB(1.66 times,P=0.0370)?IFNG(3.58 times,P=0.0020)?IRF1(1.73 times,P=0.0290)?MAP2K3(1.93 times,P=0.0099)?MAPK8IP3(2.10 times,P=0.0035)?CSF2(4.35 times,P=0.0037)?BCL2(1.90 times,P=0.0278).Three genes were down-regulated,down-regulated genes encompassed CXCL8(-3.71 times,P=0.0375)?IFNA1(-5.45 times,P=0.0025)?CXCL10(-9.44 times,P=0.0466).(2)RT-PCR verification results Four genes were randomly selected from the19 differential expression genes for RT-PCR validation.The fold change of the gene in the RT-PCR was as follows: TLR6(2.17 times,P=0.0385),TBK1(2.40 times,P=0.0414),RELA(1.94 times,P=0.0446),CXCL10(-2.46 times,P=0.0374).The resulting RT-PCR was overall consistent with the trend of the findings in gene array.?Conclusion?In conclusion,our study showed a total of 19 differentially expression genes in the 84 genes associated with TLR/MyD88 signalingpathways in peripheral blood CD4+ T lymphocytes of active stage patients with MPA.These genes include Toll-like receptors,MyD88-dependent or MyD88-independent signaling pathway,TLR negative regulation proteins,downstream pathway and target genes,apoptosis-related genes and inflammatory cytokines.This suggests that TLR signaling pathway of lymphocytes,an immune-inflammatory regulatory signal pathway,may be involved in the pathogenesis of MPA.Part ? Study on gene expression profiles of TLR/MyD88 signaling pathway related genes in peripheral blood CD8+ T lymphocytes of patients with microscopic polyangiitis?Objective?In this study,we used gene chip technology to detect mRNA expression levels of TLR/My D88-related genes in CD8+ T lymphocytes from peripheral blood of active stage patients with microscopic polyangiitis(MPA),and screened genes that were differentially expressed with healthy individuals.?Methods?A case-control study was conducted to collect 17 active stage patients with MPA(case group)and 15 healthy volunteers(control group)as subjects and collect the clinical data of the patients.CD8+ T lymphocytes were extracted from peripheral blood of all subjects.Total RNA was extracted from CD8+ T lymphocytes,and reversely transcribed in c DNA.Eight cases and six cases were randomly selected from the case group and the control group.A gene chip technology analysis was performed to study TLR/My D88-related gene m RNA expression levels.The array test of partial differential genes were verified by quantitative real-time q PCR(RT-PCR).SPSS 17.0 statistical software was used for statistical analysis.If the level of gene expression was up-regulated or down-regulated by more than 1.5 times compared with normal people,and P<0.05 was considered statistically significant.? Results ?(1)Compared with the healthy control group,23 of 84TLR/My D88-related genes were differentially expressed in the active stage patients.Five genes were up-regulated,up-regulated genes encompassed CASP8(1.82 times,P=0.0034)?IRF1(1.98 times,P=0.0057)?CIITA(2.15 times,P=0.0202)?PTGS2(3.02 times,P=0.0284)?IL10(5.71 times,P=0.0003).Eighteen genes down-regulated genes encompassed CCL2(-9.61 times,P=0.0004)?LTA(-7.19 times,P=0.0008)?CXCL10(-5.3 times,P=0.0007)?TLR9(-4.97 times,P<0.0001)?IFNB1(-4.71 times,P=0.0029)?TNF(-4.35 times,P<0.0001)?BTK(-3.80 times,P=0.0026)?MUC1(-3.79 times,P=0.0071)?CD14(-2.54 times,P=0.0127)?FADD(-2.45 times,P=0.0028)?HSPA1A(-2.41 times,P=0.0087)?NFKBIL1(-2.35 times,P<0.0001)?TLR8(-2.31 times,P=0.0218),TNFRSF1A(-1.97 times,P=0.0041)?TLR6(-1.89 times,P=0.0262)?MAPK14(-1.76 times,P=0.0131)?ELK1(-1.65 times,P=0.0014)?IRF3(-1.64 times,P=0.0144)?(2)RT-PCR verification results Four genes were randomly selected from the 23 differential expression genes for RT-PCR validation.The fold change of the gene in the RT-PCR was as follows: CXCL10(-3.90 times,P=0.0010),FADD(-3.76 times,P<0.0001),MAPK14(-1.55 times,P=0.0042),IRF1(2.12 times,P=0.0025).The resulting RT-PCR was overall consistent with the trend of the findings in gene array.?Conclusion?In conclusion,our study showed a total of 23 differentially expression genes in the 84 genes associated with TLR/My D88 signaling pathways in peripheral blood CD8+ T lymphocytes of active stage patients with MPA.These genes include toll-like receptors,TLR interacting proteins and effectors,TLR downstream pathway and target genes.This suggests that TLR signaling pathway of lymphocytes,an immune-inflammatory regulatory signal pathway,may be involved in the pathogenesis of MPA. |
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