miRNA-146a对寻常性银屑病患者外周血CD4＋T细胞的调控研究

Regulatory effects of miRNA-146a on peripheral blood CD4＋ T lymphocytes from patients with psoriasis vulgaris

目的研究miRNA-146a对寻常性银屑病患者外周血CD4’T淋巴细胞的调控，探讨miRNA-146a在寻常性银屑病发病中的作用。方法收集寻常性银屑病患者和健康对照各30例。采用实时定量PCR检测外周血CD4＋T淋巴细胞miRNA-146a表达水平，ELISA检测血浆干扰素（IFN）-y、白细胞介素（IL）-4表达水平。免疫磁珠法分选外周血CD4＋T淋巴细胞，将分离的细胞分为对照组、miRNA-146a组和miRNA-146a抑制物组，分别转染阴性对照miRNA、miRNA-146a模拟物和miRNA-146a抑制物后进行细胞培养，流式细胞仪检测Thl和Th2细胞数量，蛋白免疫印迹法和实时定量PCR分别检测IFN-1受体a（IFN-YRa）、T细胞表达T盒（T—bet）、GATA-3蛋白和mRNA的表达，ELISA检测细胞培养液上清液IFN-1、IL-4水平。结果寻常性银屑病患者外周血CD4＋T淋巴细胞miRNA-146a[（243．81±94．32）％]与血浆IFN-Y水平[（27．69±7．64）ng／L]较健康对照组[分别为（105．744-22．93）％和（9．75±2．81）ng／L]升高，差异均有统计学意义（t值分别为6．653和4．237，均P〈0．01），且miRNA-146a的表达与血清IFN-y呈正相关（r=0．837，P〈0．01）。体外CD4＋T淋巴细胞培养结果显示，与对照组比较，miRNA-146a组Th1数量、T—bet蛋白和mRNA表达、培养上清液IFN-1水平均显著增加，IFN-YRot蛋白表达降低，差异均有统计学意义（均P〈0．01）；但是，与对照组比较，miRNA-146a组和miRNA-146a抑制物组Th2细胞数量、GATA-3蛋白、GATA-3mRNA表达以及上清液中IL-4水平差异无统计学意义（均P〉0．05）。结论miRNA-146a可能通过影响Th1细胞的分化和功能参与对寻常性银屑病患者外周血CD4-T淋巴细胞的调控，在银屑病发病过程中发挥一定的作用。

Objective To evaluate regulatory effects of miRNA-146a on peripheral blood CD4~T lymphocytes from patients with psoriasis vulgaris, and to investigate the role of miRNA-146a in the pathogenesis of psoriasis vulgaris. Methods Totally, 30 patients with psoriasis vulgaris and 30 healthy human controls were enrolled into this study. Venous blood samples were obtained from these subjects, and CD4＋ T lymphocytes were isolated from these samples by using magnetic activated cell sorting （MACS）. Real-time quantitative PCR （RT-PCR） was performed to measure the expression of miRNA-146a in peripheral blood CD4＋ T lymphocytes, and enzyme-linked immunosorbent assay （ELISA） to determine plasma levels of interferon-Y（IFN-3,） and interleukin 4 （IL-4）. Some CD4＋ T lymphocytes were divided into 3 groups to be transfected with 50 nmol/L negative control miRNA （control group）, miRNA-146a mimics （miRNA-146a group） or miRNA-146a inhibitor （miRNA-146a inhibitor group）. After 24-hour additional cuhure, flow cytometry was conducted to determine the number of Thl and Th2 cells, Western-blot analysis and RT-PCR were performed to measure the protein and mRNA expressions of IFN-Y/receptor ot （IFN-YRa）, T-box expressed in T cells （T-bet） and GATA- binding protein-3 （GATA-3） respectively, and ELISA was carried out to determine the levels of IFN-Y/ and IL-4 in supernatants of CD4 ＋ T lymphocytes. Results Compared with the healthy control group, the patient group showed significantly increased miRNA-146a expression in peripheral blood CD4 ＋ T lymphocytes （243.81% ±94.32% vs. 105.74% ±22.93%, t = 6.653, P 〈 0.01 ） and plasma IFN-Y/level （27.69 ±7.64 ng/L vs. 9.75 ± 2.81 ng/L, t = 4.237, P 〈 0.01 ）. Moreover, miRNA-146a expression was positively correlated with plasma IFN-Y level in the patients （r = 0.837, P 〈 0.01 ）. After 24-hour culture in vitro, there was a significant increase in the number of Thl ceils, protein and mRNA expressions of T-bet, and supernatant level of IFN-% but a significant decrease in the protein expression of IFN-Y,RcY in the miRNA-146a group compared with the control group （all P 〈 0.01 ）. However, no significant differences were observed in the number of Th2 cells, mRNA or protein expressions of GATA-3, or supernatant level of IL-4 among the control group,miRNA-146a group and miRNA-146a inhibitor group （all P 〉 0.05）. Conclusion miRNA-146a may play an important role in the pathogenesis of psoriasis vulgaris by participating in the regulation of peripheral blood CD4＋ T lymphocytes via affecting Thl cell differentiation and function.