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目的探讨系统性红斑狼疮(systemic lupus erythematosus,SLE)患者外周血CD4细胞和CD19细胞中微小RNA(miRNA)的表达情况,及外周血CD4细胞和CD19细胞中miRNA异常表达对它们功能的影响。方法密度梯度离心法分离获取健康人及SLE患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC),磁珠分选获取CD4细胞和CD19细胞;试剂盒提取miRNA,将每组单个样本miRNA混合成总miRNA,进行人miRNA微阵列分析各个miRNA表达情况;实时荧光定量PCR(real time PCR)验证上述结果;电穿孔法转染miRNA抑制剂或模拟物至外周血CD4细胞和CD19细胞,验证miRNA对CD4细胞和CD19细胞功能的影响。结果 miRNA微阵列分析显示,与健康对照组相比,SLE患者外周血CD4细胞中5种miRNA表达升高(P<0.01),5种miRNA表达降低(P<0.01);CD19细胞中5种miRNA表达升高(P<0.01),9种miRNA表达降低(P<0.01)。Real time PCR验证结果显示,与健康对照组相比,SLE患者外周血CD4细胞中miR-126表达升高(P<0.01),miR-142-3p表达降低(P<0.01);CD19细胞中miR-200b表达升高(P<0.01),miR-17、miR-103和miR-1246表达降低(P<0.01)。进一步功能实验显示,这些异常表达的miRNA与CD4细胞及CD19细胞分泌IL-4、IL-10和IL-21及Ig G相关(P<0.01)。结论 SLE患者外周血CD4细胞和CD19细胞中存在着miRNA的异常表达,且这些miRNA可能参与SLE的发病。
Objective To investigate the expression of microRNA (miRNA) in peripheral blood mononuclear cells and CD19 cells in patients with systemic lupus erythematosus (SLE) and the effect of miRNA abnormalities in peripheral blood CD4 cells and CD19 cells on their functions. Methods Peripheral blood mononuclear cells (PBMCs) from healthy people and patients with SLE were isolated by density gradient centrifugation. CD4 and CD19 cells were sorted by magnetic beads. The miRNAs were extracted from the kit and miRNAs from each sample were mixed Total miRNAs, human miRNA microarray analysis of each miRNA expression; real-time PCR (real time PCR) to verify the above results; electroporation transfected miRNA inhibitors or mimics to peripheral blood CD4 cells and CD19 cells to verify the miRNA pairs CD4 cells and CD19 cell function. Results miRNA microarray analysis showed that the expression of five miRNAs in peripheral blood CD4 + T cells of SLE patients was significantly increased (P <0.01) and the expression of five miRNAs was significantly decreased in SLE patients compared with healthy controls (P <0.01) (P <0.01), and the expression of 9 miRNAs decreased (P <0.01). The results of Real time PCR showed that the expression of miR-126 in peripheral blood mononuclear cells was significantly increased (P <0.01) and the expression of miR-142-3p was decreased in SLE patients compared with healthy controls (P <0.01) -200b expression increased (P <0.01), miR-17, miR-103 and miR-1246 expression decreased (P <0.01). Further functional experiments showed that these abnormally expressed miRNAs were associated with the secretion of IL-4, IL-10, IL-21 and Ig G from CD4 cells and CD19 cells (P <0.01). Conclusion The abnormal expression of miRNA in peripheral blood CD4 cells and CD19 cells in SLE patients may occur and these miRNAs may be involved in the pathogenesis of SLE.