Runx3对银屑病患者Th1/Th2细胞平衡的影响及其作用机制

Effect of Runx3 on the balance of Th1 / Th2 cells in psoriasis and investigation on its mechanism

目的检测Runx3在银屑病患者和健康人外周血CD4^+T细胞中转录水平的表达,分析其对Th1/Th2细胞平衡的影响及在银屑病发病中可能的作用机制。方法选取40例银屑病患者(银屑病组)和35例健康人(健康对照组),应用密度梯度离心法分离其外周血CD4^+T细胞,采用实时荧光定量聚合酶链反应(qRT-PCR)检测Runx3转录水平的表达。将体外培养的银屑病患者的CD4^+T细胞随机分为空白对照组(未经任何处理组)、阴性对照组(对照siRNA转染组)和Runx3 siRNA组(Runx3 siRNA转染组)。采用Western blot检测沉默效果,流式细胞仪分析Th1、Th2细胞数目,酶联免疫吸附试验检测Th1和Th2细胞相关特异性分泌因子的表达水平,同时采用qRT-PCR和Western blot检测Runx3对Th1/Th2细胞转录因子表达的影响。结果银屑病组患者CD4^+T细胞Runx3 mRNA的表达水平显著高于健康对照组(P<0.01)。与阴性对照组比较,Runx3 siRNA组银屑病患者CD4^+T细胞中Runx3蛋白表达水平下降(P<0.01),同时Th1细胞数目下降(P<0.05),而Th2细胞数目上调(P<0.05),且Th1/Th2细胞比值显著降低(P<0.05);白细胞介素-2(IL-2)、干扰素-γ(IFN-γ)表达水平显著下降(P<0.01),IL-4、IL-10表达水平显著升高(P<0.05);Th1型特异性转录因子T-bet的表达显著降低(0.73±0.06 vs 0.96±0.17)(P<0.01),同时Th2型特异性转录因子GATA3的表达显著升高(1.27±0.11 vs 1.01±0.14)(P<0.05)。结论 Runx3在银屑病患者外周血CD4^+T细胞中表达上调,并且可能通过调控Th1/Th2细胞相关特异性转录因子的表达引发Th细胞失衡,从而参与银屑病的病理发病进程。

Objective To analyze the expression of Runx3 in CD4 ＋ T cells isolated from normal people and psoriasis patients, as well as its effect and the potential mechanism on the balance of Thl/Th2 cells in the pathological process of psoria- sis. Methods Forty patients with psoriasis vulgaris （psoriasis group） and 35 healthy people（ healthy control group） were en- rolled in the study. The CD4 ＋ T cells in peripheral blood were isolated by density gradient centrifugation. The expression level of Runx3 in CIM ~ T cells was analyzed by quantitative real-time polymerase chain reaction（ qRT-PCR）. The isolated cells were divided into three groups：blank control group （without any treatment） , negative control group （treated with siRNA control transfection） and Runx3 siRNA group （ transfected with Runx3 siRNA）. The transfection efficiency was evaluated by Western blotting. Flow eytometry was performed to analyze the number of Thl and Th2 cells. The specific cytokine levels of Thl and Th2 were detected by enzyme-linked immuno sorbent assay. Furthermore, the effect of Runx3 siRNA on Thl / Th2 cell tran- scription factor expression was also explored by qRT-PCR and Western blot. Results Compared with healthy control group, Runx3 protein expression in CIM ＋ T cells in psoriasis group increased significantly（P 〈 0.01 ）. Compared with negative control group, the expression of Runx3 protein in CD4 ＋ T cells of psoriasis patients in Runx3 siRNA group decreased significantly, which resulted in the decreasing of the number of Thl and increasing of the number of Th2, as well as the ratio of Thl/Th2 de- creased significantly （ P 〈 0.05 ） ; moreover, the expressions of interleukin-2 （ IL-2 ） and interferon-γ（IFN-γ） decreased signifi- cantly while the expressions of IL-4 and IL-10 increased significantly in Runx3 siRNA group（P 〈0.05） ;the expression level of Thl-specific transcription factor T-bet decreased significantly while the expression level of Th2-specific transcription factor GATA3 increased significantly in Runx3 siRNA group （ P 〈 0.05 ）. Conclusion Runx3 is up-regulated in peripheral blood CIM ＋ T cells from psoriasis patients. Importantly, Runx2 can adjust the imbalance of Thl / Th2 cells via regulating their spe- cific transcription factors,indicating a critical role in the pathological process of psoriasis.