白细胞介素35对口腔扁平苔藓患者外周血辅助性T细胞17与调节性T细胞平衡的影响

目的探讨外源性白细胞介素(interleukin,IL)35对口腔扁平苔藓(oral lichen planus,OLP)患者外周血中辅助性T细胞17(helper T cell 17,Th17)与调节性T细胞(regulatory T cell,Treg)平衡的影响。方法选取2016年10至12月就诊于贵州医科大学附属医院口腔内科黏膜专科门诊的12例OLP患者外周血(OLP组)(男性1例,女性11例,26~68岁;其中非糜烂型OLP4例,糜烂型OLP 8例),同期收集贵州医科大学附属医院体检中心的13名健康人外周血(健康对照组)(男性1名,女性12名,20~68岁),无菌提取两组外周血单个核细胞,流式细胞术(flow cytometry,FCM)分选外周血CD4+T细胞,采用实时荧光定量PCR(quantitative real-time PCR,qPCR)检测两组外周血CD4+T细胞中Th17、Treg细胞特异转录因子维甲酸相关孤核受体γt(retinoic acid receptor-related orphan receptorγt,RORγt)、叉头状转录因子(forkhead box3,Foxp3)mRNA表达水平;将OLP患者外周血分选出的CD4+T细胞分为实验组与对照组,实验组加入重组人IL-35蛋白(recombinant human IL-35,rhIL-35),对照组加入等体积磷酸盐缓冲液,分别进行细胞体外培养,收集培养结束后的细胞,qPCR检测上述因子的表达水平。结果OLP组CD4+T细胞中Foxp3、RORγt mRNA的相对表达量M(Q25,Q75)分别为0.15(0.09,0.30)和1.04(0.45,2.15)]均显著大于健康对照组分别为0.04(0.02,0.06)和0.10(0.05,0.11)](Z=-4.134,P<0.01;Z=-3.699,P<0.01)。OLP组RORγt/Foxp3 mRNA比值6.22(3.67,15.34)]显著大于健康对照组2.50(1.24,5.23)](Z=-2.665,P=0.007)。OLP患者外周血实验组CD4+T细胞Foxp3 mRNA相对表达量0.40(0.21,1.22)]显著大于对照组0.15(0.11,0.26)](Z=-2.510,P=0.012),两组RORγt mRNA表达差异无统计学意义(P>0.05),实验组RORγt/Foxp3 mRNA比值3.44(1.55,8.16)]显著小于对照组6.22(4.43,12.21)](Z=-2.746,P=0.006)。结论OLP患者外周血中存在Th17细胞占优势的Th17/Treg平衡异常,外源性IL-35可通过促进Treg细胞扩增,实现对OLP患者外周血中Th17/Treg平衡的调节。

Effects of interleukin-35 on the balance of helper T cell 17/regulatory T cell in peripheral blood of patients with oral lichen planus

Objective To investigate the effects of exogenous interleukin(IL)-35 on the balance of helper T cell 17(Th17)and regulatory T cell(Treg)in peripheral blood of patients with oral lichen planus(OLP).Methods Totally 12 peripheral blood samples of OLP patients(OLP group,one male and 11 female,26-68 years old;four cases of reticular OLP and eight cases of erosive OLP)were collected from patients of Department of Oral Mucosal Specialist of the Affiliated Hospital of Guizhou Medical University from October to December 2016.During the same period,thirteen normal peripheral blood samples were collected from the Physical Examination Center of the Affiliated Hospital of Guizhou Medical University(normal control group,one male and 12 female,20-68 years old).The peripheral blood mononuclear cells(PBMC)were extracted in sterile condition and CD4+T cells were sorted by flow cytometry(FCM).Quantitative real-time PCR(qPCR)technique was used to detect the mRNA expression levels of retinoid-related orphan nuclearγt(RORγt)and forkhead box3(Foxp3).The CD4+T cells were divided into experimental group and control group.The CD4+T cells of experimental group were cultured in vitro by adding rhIL-35,and the CD4+T cells of control group were cultured with the same volume of phosphate buffered saline(PBS).After the completion of the culture,the cells were collected.The expression levels of the same factors were detected by qPCR.Results The expressionM(Q25,Q75)]of Foxp30.15(0.09,0.30)]and RORγt mRNA1.04(0.45,2.15)]in the CD4+T cells of OLP were significantly higher than those in normal control group0.04(0.02,0.06),0.10(0.05,0.11)](Z=-4.134,P<0.01;Z=-3.699,P<0.01).The ratio of RORγt/Foxp3 mRNA in OLP group6.22(3.67,15.34)]was higher than that in normal control group2.50(1.24,5.23)](Z=-2.665,P=0.007).In the CD4+T cells of OLP patients,the expression of Foxp3 mRNA in the experiment group0.40(0.21,1.22)]was higher than that in the control group0.15(0.11,0.26)](Z=-2.510,P=0.012),and the expression of RORγt mRNA between two groups showed no significant difference(P>0.05).The RORγt/Foxp3 mRNA ratio3.44(1.55,8.16)]of the experiment group was lower than that in the control group6.22(4.43,12.21)](Z=-2.746,P=0.006).Conclusions There was a Th17/Treg imbalance with predominated by Th17 cells in the peripheral blood of patients with OLP.Exogenous rhIL-35 had an immunomodulatory effect on the balance of Th17/Treg.