药物特异性T细胞在重症药疹发病机制中的作用研究

发布时间：2018-04-06 22:25

本文选题：重症药疹　切入点：药物特异性T细胞　出处：《第四军医大学》2010年硕士论文

【摘要】： 药疹是一种由药物引起的不良反应。药物通过口服、注射或吸入等途径,进入人体内而引发的皮肤或粘膜的炎症性损害。重症药疹是皮肤科常见急症,其种类繁多,Stevens- Johnson综合征(Stevens-Johnson Syndrome;SJS)、大疱性表皮坏死松解型药疹(Toxic Epidermal Necrolysis;TEN)是其中最严重的两种类型,其病情变化快,皮疹广泛,常伴有高热,严重者会出现全身症状及肝肾功能紊乱,此外还伴有大面积表皮内大疱,以及皮肤和粘膜的剥脱,具有较高的死亡率。药疹的发病机制尚不明确,近年来,越来越多的研究发现重症药疹的发病与免疫学机制有密切的关系。在对重症药疹的研究中,国外相继发现:①SJS与TEN患者在首次发生药物过敏反应之后,再次接触致敏药物反应更迅速,也更强烈,对患者机体的损伤也更为严重;②HLA基因在决定患者是否罹患重症药疹具有重要作用,HLA-B*1502与卡马西平、HLA-B*5801与别嘌呤醇导致的重症药疹相关;③重症药疹患者皮损、水疱内含有HLA限制性、具有细胞毒活性的CD8+ T淋巴细胞;④应用药物及其衍生物,可从患者外周血单核细胞(peripheral blood monouclear cells;PBMC)中培养获得药物特异性T细胞株和T细胞克隆。上述结果证实免疫学机制在SJS、TEN发病中具有关键作用。大多数发疹型药疹的发病与细胞免疫密切相关,属于Th1反应优势,IFN-γ是Th1途径释放出的主要因子,重症药疹也不例外,患者外周血T细胞,受到致敏药物刺激后,Th1释放IFN-γ增加。 Fas系统包括Fas、FasL、可溶性FasL。FasL与Fas结合后能诱导表达Fas的细胞凋亡;Fas及其配体FasL是近年来研究得最为深入的有关细胞凋亡的膜表面分子。有学者通过FasL激活Fas是导致TEN患者表皮细胞凋亡的第一步。在所有SJS、TEN患者血清内的sFasL浓度是明显升高的,而多形红斑型药疹及正常人中sFasL没有明显升高。但SJS、TEN之间sFasL并没有显著差异。临床发病过程分析表明,SJS、TEN患者血清内的sFasL,会随着时间增加而逐渐减少。在重症药疹发病过程中,有些病人在发病过程中会产生大小不一的松弛型水疱、大疱,受外力后可形成糜烂面,皮损免疫病理显示表皮内以CD8+细胞占优势,而真皮内细胞浸润则以CD4+细胞占优势。因此我们决定以T细胞功能为研究对象,通过观察T细胞功能与药物刺激的关系,体外检测IFN-γ、sFasL分泌水平,不仅可以证明经致敏药物刺激后的T细胞可分泌炎症因子,从而引起了表皮细胞的损伤;也将有可能应用于临床上重症药疹患者致敏药物的鉴定研究,为临床医师提供可靠的患者致敏药物判定依据。目的: 以重症药疹患者药物特异性T细胞的细胞因子及细胞毒蛋白为研究对象,研究重症药疹药物致敏及损伤表皮的机制,为建立体外致敏药物检测方法和开展重症药疹的临床救治工作打下基础。方法: 共收集10例重症药疹患者(住院患者和治愈后门诊随访患者),抽取患者外周血分离单个核细胞(PBMC),经相应的致敏药物刺激后培养出T细胞株,用ex vivo ELISPOT和Cultured ELISPOT的方法检测患者PBMC及药物特异性T细胞株分泌干扰素-γ(interferon-γ;IFN-γ)的情况。对10例患者同时设立与致敏药物分子结构不同的无关药物对照组。将经致敏药物刺激后药物刺激孔上清与HaCaT细胞共培养18h。用光镜、荧光显微镜、流式细胞仪检测HaCaT细胞凋亡的情况;以及用ELISA的方法,观察重症药疹患者药物特异性T细胞株上清液中sFasL含量。其中重症药疹患者与正常人各10例;经检验,其流行病学特征与总体样本无统计学差异;二组年龄差异无统计学意义(p0.05),具有可比性。结果: 10例重症药疹患者PBMC经致敏药物刺激后IFN-γ的分泌均明显高于对照药物组、正常人对照组及其他类型药疹组(P0.001)。10例其他类型药疹中有四例患者PBMC经致敏药物刺激后IFN-γ的分泌较对照药物及正常人对照组均略有升高(P0.05)。另外IFN-γ的分泌会随着与致敏药物共培养天数的增加而增多。使用相应致敏药物培养出药物特异性的T细胞株,并采用ELISPOT的方法观察后发现,药物特异性T细胞株经致敏药物刺激后IFN-γ的分泌明显高于正常人对照及非重症药疹组(P0.001)。10例其他类型药疹中有四例患者T细胞株经致敏药物刺激后IFN-γ的分泌较对照药物组及正常人对照组有所升高(P0.05)。SNK-q检验分析结果显示,PBMC、T细胞株两组间IFN-γ的分泌有显著差异(P0.05),经药物刺激后的T细胞株IFN-γ的分泌水平高于PBMC组。为证明药物特异性T细胞在患者体内长期存在,我们在10例患者中寻找到3例病史为1-3年的患者,同样发现其PBMC及T细胞株中均存在药物特异性T细胞株。另外,用光镜、荧光显微镜、流式细胞仪检测中均可以看到加入2%药物刺激孔上清组,HaCat凋亡明显多于1%药物刺激孔上清组;而加入抗FasL的抗体anti-FasL mAb(1μg/mL)后,HaCat细胞凋亡减少。并且,重症药疹患者药物特异性T细胞株上清液中sFasL含量,明显高于正常人对照组(P0.01)。结论: 重症药疹患者体内存在着药物特异性T细胞。ex vivo ELISPOT联合Cultured ELISPOT方法在体外检测患者PBMC、T细胞株经致敏药物刺激后产生IFN-γ的分泌水平,可能有助于致敏药物的鉴定。药疹患者治愈后体内持续存在药物特异性的T细胞,这提示患者应严格避免致敏药物是的再次服用。外周血中单个核细胞分泌大量死亡受体的配体sFasL与角质细胞中的Fas相结合,从而导致角质形成细胞的凋亡;而阻断sFasL后角质形成细胞凋亡明显减少,从而说明角质形成细胞凋亡过程中发挥着重要作用。
[Abstract]:Drug eruption is a drug - induced adverse reaction . Drugs enter into human body through oral , injection or inhalation route . Severe drug eruption is one of the most serious types . Stevens - Johnson Syndrome ( SJS ) , bullous epidermal necrolysis type ( Stevens - Johnson Syndrome ; SJS ) , bullous epidermal necrolysis type ( SJS ) , bullous epidermal necrolysis type ( SJS ) , bullous epidermal necrolysis type ( SJS ) , bullous epidermal necrolysis type ( SJS ) , bullous epidermal necrolysis type drug eruption ( TEN ) are the most serious types , with severe symptoms and disorder of liver and kidney function .

The pathogenesis of drug eruption is not clear . In recent years , more and more studies have found that the pathogenesis of severe drug eruption is closely related to immunological mechanism .

Most of the rash type drug eruption is closely related to cellular immunity , which belongs to Th1 response advantage . IFN - 纬 is the main factor released by Th1 pathway , and the severe drug eruption is not the exception , and the peripheral blood T cell of the patient is stimulated by the sensitizing drug , and the Th1 release IFN - 纬 increases .

The expression of Fas , FasL , soluble FasL , FasL and Fas combined with Fas could induce the apoptosis of apoptotic cells . Fas and FasL were the first step in the study of apoptosis in TEN patients . The expression of sFasL in serum of all SJS and TEN patients was significantly higher than that in normal controls . However , the sFasL levels in serum of SJS and TEN were not significantly different .

In the course of the onset of severe drug eruption , some patients may develop a loose blister or blister during the course of the disease , which can form erosion surface after external force , and the immune and pathological changes of skin lesions show that CD8 + cells are dominant in the epidermis , while the infiltration of the cells in the dermis is dominated by CD4 + cells .

Therefore , we decided to study the relationship between T cell function and drug stimulation by observing the relationship between T cell function and drug stimulation , and detect the secretion of IFN - 纬 and sFasL in vitro .

Purpose :

In order to establish an in vitro drug - sensitive drug detection method and to carry out the clinical treatment of severe drug eruption , we study the mechanism of drug - specific T - cell and cytotoxic protein of drug - specific T cell in severe drug eruption .

Method :

A total of 10 patients with severe drug eruption ( hospitalized patients and post - cured outpatient follow - up patients ) were collected , and mononuclear cells ( PBMC ) were isolated from peripheral blood of patients . T cell lines were cultured after stimulation with corresponding sensitizing drugs , and IFN - 纬 ( IFN - 纬 ) secretion of PBMC and drug - specific T cell lines was detected by ex vivo ELISA .

The levels of sFasL in the supernatant of drug - specific T cell lines in patients with severe drug eruption were examined by light microscopy , fluorescence microscope and flow cytometry , and the levels of sFasL in the supernatant of drug - specific T cell lines were observed by ELISA .

Results :

IFN - 纬 secretion in PBMCs of 10 patients with severe drug eruption was significantly higher than that of control drug group , normal control group and other types of drug eruption group ( P0.001 ) . The secretion of IFN - 纬 increased slightly in four patients with other types of drug eruption ( P0.05 ) .

The secretion of IFN - 纬 was significantly higher than that of control group and non - serious drug eruption group ( P0.05 ) .

In order to demonstrate the long - term presence of drug - specific T cells in patients , we found 3 patients with a history of 1 - 3 years in 10 patients , and also found that there were drug - specific T cell lines in both PBMC and T cell lines .

In addition , the apoptosis of HaCat cells was significantly higher than that of normal controls ( P0.01 ) .

Conclusion :

In patients with severe drug eruption , there is a drug - specific T - cell in the body of patients with severe drug eruption .

The ligand sFasL secreting a large number of death receptors in peripheral blood is combined with Fas in keratinocytes , which leads to the apoptosis of keratinocytes . The apoptosis of keratinocytes after blocking the sFasL is significantly reduced , which indicates that the keratinocytes play an important role in the process of apoptosis .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R758.25

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