银屑病调节性T细胞的功能异常及STAT3通路调控机制研究

Dysfunction of regulatory T cells in patients with psoriasis and related mechanisms of regulation by the STAT3signaling pathway

目的研究银屑病患者外周血调节性T细胞（Treg）的功能，探讨与功能异常相关的STAT3信号通路机制。方法寻常性银屑病患者81例，银屑病面积和严重度指数（PASI）评分10～30，均为慢性斑块状。对照组46例，为健康献血者。采用流式细胞仪检测外周血中Treg细胞的比例，用体外淋巴细胞混合培养方法检测银屑病患者和健康人外周血中Treg细胞的增殖活性及对效应性T细胞（Tresp）的抑制功能，用流式细胞仪及qRT—PCR检测Treg细胞中磷酸化STAT3的比例及分泌促炎因子干扰素γ（IFN-γ）、肿瘤坏死因子a（TNF—a）、白细胞介素17（IL-17）的水平。最后，用STAT3通路抑制剂StatticV处理银屑病患者Treg细胞，观察其增殖及抑制功能的恢复及分泌促炎因子的变化。结果银屑病患者组外周血Treg细胞数量（6．437％±0．186％）与对照组（6．812％±0．241％）比较差异无统计学意义（t=1．224，P〉0．05），但银屑病患者组外周血Treg细胞增殖活性及对Tresp细胞的抑制功能明显降低，磷酸化STAT3表达水平显著升高，分泌促炎因子IFN-γ、TNF—d、IL-17的水平显著升高（均P〈0．05）。经50肌蜀几StatticV作用后，银屑病患者Treg细胞对Tresp抑制率为61．670％±4．640％，未处理组为28．820％±11．490％，两组差异有统计学意义（P〈0．05）；50μg／LStatticV作用后，促炎因子IFN-ⅥTNF-a、IL-17mRNA表达量（2-△△Q）分别为1．654±0．879、0．850±0．705、0．572±O．135，均显著低于未处理组（分别为23．350±6．721、4．847±1．525、3．095±0．650），差异均有统计学意义（P〈0．05）。结论银屑病患者Treg细胞对Tresp细胞的负向调控功能降低，其机制与STAT3信号通路异常活化有关，抑制STAT3通路的活化有可能一定程度地恢复Treg细胞功能。

Objective To evaluate the function of regulatory T （Treg） cells in peripheral blood from patients with psoriasis, and to explore the possible role of the STAT3 signaling pathway in Treg cell dysfunction. Methods Totally, 81 patients with psoriasis vulgaris, who all presented with chronic plaques and had a psoriasis area and severity index （PASI） score of 10 - 30, were enrolled into this study. Forty-six healthy blood donors served as the control group. Venous blood samples were collected from these subjects followed by isolation of Treg cells and responder T （Tresp） cells. Flow eytometry was performed to determine the proportion of Treg cells in peripheral blood as well as that of cells secreting phosphorylated-STAT3 （ p-STAT3 ）, interferon γ（IFN-γ）, tumor necrosis factor et （TNF-a） and interleukin 17 （ IL- 17 ） in Treg cells, and quantitative real-time PCR （qRT-PCR） to measure the expression levels of IFN-Y TNF-a and IL-17 mRNAs in Treg cells. Some Treg cells and Tresp cells were cultured in vitro alone or in combination, and flow eytometry was conducted to estimate cellular proliferative activity on day 7 after stimulation with IL-2. Some patient-derived Treg cells were classified into several groups to be cultured alone or in combination with Tresp cells with or without the presence of the STAT3 pathway inhibitor, Stattic V （10 or 50 μg/L）, for 7 days. Subsequently, flow eytometry was performed to evaluate the proliferative activity of Tresp cells, and qRT-PCR to measure the expression levels of IFN-Y TNF-a and IL-17 mRNAs in Treg cells. Results No significant differences were observed in the proportion of Treg cells in peripheral blood between the patient group and control group （6.437% ±0.186% vs. 6.812% ± 0.241%, t = 1.224, P〉 0.05）. Compared with control-derived Treg cells, the patient-derived Treg cells showed significantly decreased proliferative activity and inhibitory effects on Tresp cells, but increased proportion of cells secreting p-STAT3, IFN-% TNF-a and IL-17 （all P 〈 0.05 ）. After the treatment with 50 g/L Stattic V, a significant increase was observed in theinhibitory effect of patient-derived Treg cells on Tresp cells （inhibition rate： 61.670% ＋ 4.640% vs. 28.820% ＋ 11.490%, P〈 0.05）, but a significant decrease in the mRNA expressions of IFN-Y （2-△△Q： 1.654 ± 0.879 vs. 23.350 ± 6.721, P〈 0.05）, TNF-c~ （0.850 ± 0.705 vs. 4.847 ± 1.525, P〈 0.05） and IL-17 （0.572 ~ 0.135 vs. 3.095 ~ 0.650, all P〈 0.05） in patient-derived Treg cells compared with untreated patient-derived Treg cells. Conehmions The negative regulatory effect of Treg cells on Tresp cells is decreased in patients with psoriasis, which may be associated with abnormal activation of the STAT3 signaling pathway, and inhibition of the pathway may restore the function of Treg cells to a certain extent.