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。     

转化生长因子β受体在口腔扁平苔藓CD8+T细胞中的表达

目的 研究转化生长因子β受体(transforming growth factor-β receptor,TGF-βR)在口腔扁平苔藓(oral lichen planus,OLP)组织中CD8+T细胞中的表达,探讨TGF-βR在OLP发病中的作用.方法 采用免疫组化双标记法检测28例OLP组织中CD8+T细胞TGF-βR Ⅰ和TGF-βR Ⅱ蛋白的表达.以10例临床正常口腔黏膜(normal oral mucosa,NOM)为对照组.结果 OLP组织中CD8+及TGF-βR Ⅰ、CD8+及TGF-βR Ⅱ双标记细胞的阳性率分别为(8.82±9.98)%、(1.11±2.94)%.NOM组织中双标记细胞均为零.OLP组CD8+T细胞中TGF-βRⅡ表达与NOM组相比差异无统计学意义(P＞0.05),TGF-βR Ⅰ与NOM组相比表达增强.结论 提示OLP组织CD8+T细胞存在TGF-β信号传导异常.这可能是CD8+T细胞缺乏有效抑制、致使OLP慢性炎性反应长期持续的因素之一.     

LC3B在口腔扁平苔藓组织及其T细胞中的表达

目的:检测微管相关蛋白1轻链3B(LC3B)在口腔扁平苔藓(oral lichen planus, OLP)局部组织及其T细胞中的表达。方法:分别采用免疫组织化学染色和多色免疫荧光染色方法检测自噬相关蛋白LC3B在OLP局部组织及其T细胞中的表达,并分析LC3B与OLP临床特征之间的关系。结果:LC3B在OLP局部组织及病损T细胞中的表达显著高于对照组(P<0.001,P<0.01),且与RAE计分呈负相关(P<0.05,P<0.01)。LC3B在非糜烂型OLP组织中的表达显著高于糜烂型OLP(P<0.01)。结论:自噬相关蛋白LC3B可能参与了OLP的局部免疫机制。     

口腔扁平苔藓固有层淋巴细胞中Fas及Fas配体的表达变化

目的 检测Fas及Fas配体(Fas ligand,FasL)在口腔扁平苔藓(oral lichen planus,OLP)固有层淋巴细胞中的蛋白表达,探讨Fas、FasL和活化诱导的细胞死亡(activation-induced cell death,AICD)与OLP发病的关系.方法 采用脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记法检测31例OLP和10例正常口腔黏膜(normal oral mucosa,NOM)中淋巴细胞凋亡情况;分别使用免疫组化法检测组织总淋巴细胞及CD8~+、CD4~+T细胞Fas、FasL的表达.结果 OLP与NOM固有层淋巴细胞凋亡率[分别为(1.9±1.8)%、(11.5±9.0)%]差异有统计学意义(P=0.013).OLP淋巴细胞Fas、FasL表达与NOM组相比明显增强[阳性表达率分别为52%(16/31)、71%(22/31),P值分别为0.005、0.000].OLP中CD8~+与Fas~+细胞双阳性表达率为10%,与NOM组相比无明显升高(P=0.313),而CD8~+与FasL~+细胞双阳性表达率[58%(3/31)]显著升高(P=0.002).CD4~+与Fas~+细胞双阳性表达率为35%(11/31),较NOM组显著升高(P=0.031),其中网纹型的表达明显升高(阳性表达为8/19,P=0.019),但糜烂、萎缩型的表达无显著升高(阳性表达为3/12,P=0.097).CD4~+与FasL~+表达率为16%(5/31),与NOM组相比无明显增强(P=0.182).结论 OLP淋巴细胞凋亡低下;OLP中T细胞亚群Fas、FasL表达不均衡,CD8~+细胞和萎缩、糜烂型OLP中CD4~+细胞可能逃逸AICD,与炎症的持续和进展有关;网纹型OLP中部分CD4~+T细胞可能经历AICD.     

转录因子RORγτ和FOXP3在口腔扁平苔藓病损组织中的表达及意义

目的:口腔扁平苔藓(oral lichen planus,OLP)是一种发生于口腔黏膜的T淋巴细胞介导的自身免疫性疾病。辅助性T细胞(helper T lymphocytes, Th)在OLP的发病过程中具有重要作用,本研究旨在进一步探索OLP患者局部病损组织中辅助性T细胞亚群Th17细胞和Treg细胞的作用。方法:纳入43例OLP患者和13例健康志愿者。采用实时定量PCR法检测局部病损组织中Th17和Treg细胞的特征性转录因子RORγτ和FOXP3的表达,采用GraphPad Prism 5 软件对其表达差异进行统计学分析。结果:OLP局部病损组织中转录因子RORγτ和FOXP3的表达显著高于正常黏膜组织,而且均与OLP的临床分型密切相关。萎缩糜烂型OLP组病损组织中RORγτ/FOXP3比值显著高于网状型OLP组和健康对照组,而网状型OLP组的RORγτ/FOXP3比值虽然高于对照组,但差异无显著性。结论:Th17细胞和Treg细胞均参与OLP的局部免疫反应;同时,Th17/Treg失衡也参与了重症型OLP的致病过程,且表现为Th17细胞优势。     

口腔扁平苔藓患者外周血调节性T细胞水平的变化

目的:研究口腔扁平苔藓(OLP)患者外周血调节性T细胞(Treg)水平的改变.方法:收集口腔扁平苔藓(普通型)患者和健康人外周血.采用流式细胞术分析其中CD4+ CD25+T细胞、CD4+ CD25highT细胞、CD4+ Foxp3+ T细胞、CD4+CD25+ Foxp3+ T细胞比例的改变.结果:OLP患者外周血中CD4+ CD25high T 细胞、CD4+ Foxp3+ T细胞水平较正常对照组明显升高(P<0.05);CD4+ CD25hight细胞与CD4+ Foxp3+ T细胞水平存在正相关性(r=0.349,P<0.05),CD4+ CD25+ Foxp3+ T细胞与CD4+ Foxp3+ T细胞水平存在明显正相关性(r:0.802,P<0.01).结论:OLP患者外周血调节性T细胞水平发生了改变.     

口腔扁平苔藓患者中医证型与免疫状态的相关研究

目的 通过观察不同证型口腔扁平苔藓( lichen planus,OLP)患者的全身免疫状况,探讨中医证型与免疫的关系,以期为临床判断OLP患者免疫状况提供一种新思路.方法 纳入70例OLP患者,分为心脾蕴热型、肝经实火型、虚火上炎型和气血两虚型,所有患者均行免疫检查并对结果进行统计学分析.结果 在OLP患者中虚火上炎型37例,所占的比例最大[53％ (37/70)];OLP患者的外周血细胞总体表现为CD-3、CD-4与正常参考值相比均显著降低(P＜0.05),降低比例分别为76％ (53/70)和63％( 44/70);与正常参考值相比,心脾蕴热型组CD-3 (55.44±5.57)、CD-8(15.60±3.93)均显著降低(P＜0.05),CD-4/CD-8( 2.40±0.69)显著升高(P＜0.05),IgM(2279.33±729.23)显著升高(P＜0.05);虚火上炎型组CD-4( 30.52±7.50)显著降低(P＜0.05),IgA(2608.70±728.59)显著升高(P＜0.05);气血两虚型组CD-3( 56.10±3.24)显著降低(P＜0.05).结论 OLP患者的全身免疫状态以细胞免疫紊乱为主;不同中医证型的OLP患者其免疫状况存在一定差异性.     

口腔扁平苔藓中乳腺珠蛋白表达变化的初步研究

目的 从转录、蛋白水平验证口腔扁平苔藓(oral lichen planus,OLP)病损差异表达基因乳腺珠蛋白(secretoglobin family 2A member 2,SCGB2 A2)的变化,探讨SCGB2A2在OLP发病中的作用.方法 分别应用实时荧光定量反转录PCR、蛋白质印迹法及免疫组织化学等方法检测16例OLP病损(OLP组)和10例正常对照(正常对照组)中SCGB2A2转录水平及蛋白表达情况,采用单因素方差分析对组间结果进行比较,以双侧P＜0.05为差异有统计学意义.结果 OLP组SCGB2A2蛋白水平(1.02±0.11)显著高于正常对照组(0.48 ±0.09),两组差异有统计学意义(P=0.002);OLP组SCGB2A2的转录水平[M(Q1,Q3)][1.68(1.60,1.83)]则显著低于正常对照组[6.32 (5.24,10.87)],两组差异有统计学意义(P＜0.05).结论 SCGB2A2参与了OLP的发病,其作用机制尚有待进一步研究.     

口腔扁平苔藓合并2型糖尿病的临床及病理特征研究

目的 通过病例对照研究分析口腔扁平苔藓(oral lichen planus, OLP)合并2型糖尿病(type 2 diabetes mellitus, T2DM)的临床及组织病理学表现特征。方法 将104例患者分为OLP组与OLP+T2DM组。收集患者的基本信息,OLP的临床类型与REU评分,两位病理专家对组织病理表现进行具体描述。应用SPSS 24.0对两组间的临床与组织病理表现进行统计分析。结果 OLP+T2DM组糜烂型OLP的发生率与REU评分均显著高于OLP组(P<0.001)。随访1个月后,服用降糖药物(P<0.001)和血糖控制理想(P<0.01)的OLP患者REU评分均显著降低。多因素二元Logistic回归分析显示：高REU评分、糜烂型、淋巴细胞浸润密集、淋巴浸润带不清晰、基底层重度炎症、基底层纤维蛋白的存在(OR=1.58、37.15、8.49、10.16、3.94、4.35)是OLP合并T2DM的典型特征。结论 合并T2DM的OLP患者临床病损更严重,组织破坏程度更高,临床诊治应制定个性化方案,以获得精准治疗效果。     

口腔扁平苔藓外周血中辅助性T细胞/调节性T细胞平衡与临床特征相关性分析

目的 了解口腔扁平苔藓（OLP）患者外周血中辅助性T细胞17（Th17）与调节性T细胞（Treg）的平衡变化,探讨它们在OLP发病机制中的作用及意义。方法 选取17例正常组和33例OLP患者（网纹型15例,糜烂型18例）的外周血,应用流式细胞术（FCM）检测Th17、Treg细胞的表达水平,实时荧光定量聚合酶链反应（qPCR）检测它们的转录因子维甲酸相关孤核受体γt（RORγt）和叉头状转录因子3（Foxp3）mRNA的表达。结果 OLP外周血中Th17、Treg细胞及RORγt、Foxp3 mRNA表达均升高（P<0.05）,但Treg细胞和Foxp3 mRNA表达在OLP两型间差异无统计学意义。Th17/Treg比值在OLP中升高（P<0.05）,其中糜烂型OLP显著高于正常组及网纹型OLP（P<0.01）,但网纹型OLP与正常组相比差异无统计学意义。Spearman相关分析显示Th17细胞和Th17/Treg比值与体征计分、RAE计分存在正相关关系（r=0.66,P=0.00;r=0.66,P=0.00;r=0.52,P=0.00;r=0.50,P=0.00）;同时Th17细胞与Treg细胞也存在正相关关系（r=0.39,P=0.03）。结论 OLP外周血中Th17和Treg细胞以及它们的比例均增高,Th17/Treg失衡在糜烂型OLP的发病过程中起了一定作用。     

调节性T细胞和Th17细胞在OLP中的表达

目的：通过观察调节性T细胞（regulatory Tcells,Treg）和Thl7细胞（helper Tcells17,Th17）在口腔扁平苔藓（OLP）组织中的表达情况,探讨其在OLP发生发展过程中的变化。方法：应用免疫组织化学双标记技术检测35例OLP患者和19例对照组口腔黏膜组织中CD25＋Foxp3＋、CD4＋IL-17＋细胞的表达。结果：OLP组病损组织中有大量CD25＋Foxp3＋细胞浸润,较对照组显著增多,两者差异非常显著（P〈0．01）。CD4＋IL-17＋细胞在OLP组织中的表达较正常口腔黏膜组织有增加趋势,但差异无统计学意义（P〉0．05）。经进一步统计学分析,CD25＋Foxp3＋细胞和CD4＋IL-17＋细胞数量改变在OLP组织中表达呈正相关（P〈0．05）。结论：Treg细胞的数量在OLP组织中增多,Thl7细胞可能协同Treg细胞,在OLP发生发展过程中发挥了一定的作用。     

Overexpression and varied clinical significance of Th9 versus Th17 cells in distinct subtypes of oral lichen planus

ObjectiveOral lichen planus (OLP) presents with large numbers of T lymphocytes accumulating beneath the epithelium of the oral mucosa; however, its aetiology remains obscure. A potential role for an emerging novel T cell subset, Th9, in OLP has recently been suggested but remains to be clarified. The current aim was to investigate the expression and potential clinical significance of Th9 cells in distinct subtypes of OLP.Materials and methodsPeripheral blood samples were collected from 41 OLP patients and 18 healthy controls (HCs). Flow cytometric analysis was used to detect the CD4+ T helper subset Th9 (IL-9⁺IL-17⁻CD4⁺ Th cells) and Th17 (IL-9⁻IL-17⁺CD4⁺ Th cells) expression levels.ResultsFlow cytometry results showed significantly elevated levels of Th9 cells in reticular and erosive OLP compared to HCs. Th9 expression in erosive OLP was less than in reticular OLP, indicating that Th9 but not Th17 cells may play a predominant role in reticular disease. However, in erosive OLP patients, we found much higher levels of Th17 cells compared to reticular OLP patients and HCs, indicating that Th17 dominates in erosive OLP. Statistical analysis showed positive correlations of Th9 cells and Th17 cells in patients with reticular or erosive OLP but none in HCs.ConclusionsTh9 and Th17 cells may take the predominant roles in reticular and erosive OLP respectively, and their numbers were positively correlated in reticular and erosive OLP patients. Elevated circulating Th9 cells may help maintain immune balance in OLP immunopathogenesis, which requires further investigation.     

T细胞免疫和细胞凋亡在口腔扁平苔藓发病中的作用

目的通过探讨口腔扁平苔藓（OLP）中细胞凋亡情况及CD4＋、CD8＋T细胞和CD4/CD8比例的变化分析细胞免疫与细胞凋亡的关系,进一步了解OLP的发病机制。方法应用免疫组化SP法检测27例OLP组织中CD4＋、CD8＋T细胞的表达水平,并用原位末端转移酶标记法（TUNEL）定位检测17例OLP中细胞凋亡情况。结果OLP组固有层中CD4＋、CD8＋T细胞明显高于对照组（P＜0.05）,CD4/CD8值降低。OLP组中上皮内细胞凋亡指数高于对照组,固有层淋巴细胞凋亡指数明显低于对照组。结论OLP组固有层中CD4＋、CD8＋T细胞浸润的增加、CD4/CD8比值的变化及OLP中上皮细胞和固有层淋巴细胞凋亡异常,说明细胞免疫功能紊乱和细胞凋亡异常在OLP发病中起重要作用。     

Cellular immune correlates of clinical severity in oral lichen planus: preliminary association with mood states

OBJECTIVES: We investigated cellular immune and psycho-immune dysfunctions in patients with erosive and non-erosive oral lichen planus (OLP) lesions.
METHODS: Patients with erosive or non-erosive OLP were screened at the UCLA Dental Clinic. The profile of mood states (POMS) was administered. T lymphocyte subpopulations were monitored by dual fluorescence. T lymphocytes were stimulated with phytohemagglutinin (PHA) for assessment of markers of activation by flow cytometry and of interleukin (IL)-2 production by ELISA.Plasma cortisol and neopterin levels were assessed by radioimmunoassay.
RESULTS: Circulating T cells that express the cluster of differentiation no.4 (CD4⁺) but devoid of the CD45RA marker, and POMS score were significantly associated ( r = 0.83, P < 0.05) in the patients we studied. We found a significantly higher ( P < 0.05) per cent and absolute lymphocyte numbers of circulating CD4+CD45RA cells in the OLP patients with erosive lesions, compared to OLP patients with non-erosive lesions. The ratio of CD4⁺ CD45RA⁺ over CD4+CD45RA cells was significantly ( P < 0.05) biased toward the CD4+CD45RA subpopulation in OLP patients with erosive lesions (ratio = 0.19 ± 0.09) compared to patients with non-erosive OLP lesions (ratio = 0.47 ± 0.15). The expression of CD54, but not that of CD69, was significantly blunted ( P < 0.05) in OLP patients following CD3⁺ cell stimulation. IL-2 production and plasma neopterin were normal in these patients. There was no correlation between plasma cortisol and T cell populationS. CONCLUSIONS: We find fine differences in psycho-immune interactions between patients afflicted with non-erosive OLP lesions compared to those with erosive OLP lesions.     

OLP外周血中CD4＋CD25＋T细胞体外增殖及对CD4＋CD25-T细胞增殖的影响

目的：通过检测口腔扁平苔藓（OLP）患者外周血中CD4＋CD25＋T细胞的体外增殖及对CD4＋CD25＋T细胞增殖的影响,探讨调节性T细胞对OLP特异性细胞免疫的调节作用及其在该病发生中的意义。方法：通过免疫磁珠分离系统（MACS）分选OLP患者和健康志愿者外周血单个核细胞（peripheral blood mononuclear cell,PBMC）中的调节性T细胞（regulatory Tcell,Treg）和效应性T细胞（responder Tcell,Tresp）,采用流式细胞术检测其纯度、3H--脱氧胸腺嘧啶苷方法检测CD4＋CD25＋T细胞对CD4＋CD25＋T细胞增殖的影响,比较OLP患者和健康志愿者体内Treg细胞功能。结果：分选后健康对照组及OLP患者外周血中CD4＋CD25＋T细胞纯度均高于85％。无论是健康对照还是OLP患者的CD4＋CD25-T细胞均明显抑制CD4＋CD25-T细胞的增殖。与健康对照组相比,OLP组CD4＋CD25＋T细胞对CD4＋CD25-T细胞增殖的抑制作用显著降低旧〈0．01）。结论：调节性T细胞可能通过抑制OLP特异性细胞免疫应答促进疾病的发生发展。     

FOXP3+ T regulatory cells in lesions of oral lichen planus correlated with disease activity

Objective:  The aim of this study was to determine the correlation between the number of FOXP3⁺ T cell in lesions and the disease activity of patients with oral lichen planus (OLP).
Materials and Methods:  The expression of FOXP3 was investigated using immunohistochemical staining and real-time RT-PCR in 23 OLP lesions and 12 controls. Changes of FOXP3⁺ Treg in peripheral blood from three patients' pre and post-treatment were assessed using flow cytometry.
Results:  Few FOXP3⁺ cells were detected in controls, but an increased number of FOXP3⁺ cells were observed in lesions ( n  = 20, 40.99 ± 24.68 cells per high-power field – hpf). Furthermore, the frequency of FOXP3⁺ Treg in reticular OLP ( n  = 7, 63.6 ± 23.2 cells per hpf) was significantly higher than that in erythematous/erosive OLP ( n  = 13, 28.8 ± 16.8 cells per hpf, P  = 0.001). In addition, negative correlation was found between the number of FOXP3⁺ Treg and disease activity (correlation oefficient = −0.557, P  = 0.013). The proportion of FOXP3⁺ Treg showed remarkable increase in peripheral blood from patients after treatment (1.39 ± 0.71% vs 4.91 ± 1.59%).
Conclusions:  These data indicated that FOXP3⁺ Treg were involved in the pathogenesis of OLP and correlated with disease's subtype and activity.     

口腔扁平苔藓患者Th1／Th2失衡表达的研究

目的：观察口腔扁平苔藓（oral liclaen planus,OLP）患者外周血中Th1、Th2型细胞转录因子T-het和GATA-3以及Thl、Th2型细胞因子的表达,进一步探索OLP的发病机制。方法：通过密度梯度离心法分离20例充血糜烂型,16例光滑型OLP病例和19例正常对照组人群的外周血单个核细胞,逆转录-聚合酶链反应（RT—PCR）法检测各组外周血单个核细胞中T—betmRNA和GATA-3 mRNA的表达;ELISA法检测各组血清中干扰素-γ（interferon gamma,IFN-1）和白细胞介素-4（interleukin-4,IL-4）的表达。结果：充血糜烂型和光滑型OLP患者中T—betmRNA、IFN-γ的表达均低于正常对照组,差异有统计学意义（P〈0．01）;而充血糜烂型和光滑型OEP患者中GATA-3 mRNA、IL-4的表达均高于正常对照组,差异有统计学意义（P〈0．01）。T—bet mRNA和GATA-3 mRNA的表达在充血糜烂型及光滑型OLP患者组间的差异有统计学意义（P〈0．01）;而IFN-γ和IL-4的表达在充血糜烂型及光滑型OLP患者组间的差异无统计学意义（P〉0．05）。结论：Th1／Th2失衡表达与OLP发病机制密切相关,为临床治疗OLP提供参考。