醋甲唑胺与HLA-B*59:01分子相互作用诱发超敏反应导致SJS/TEN的机制研究

Cutaneous adverse drug reactions (CADRs) compose a significant proportion of all adverse drug reactions (ADRs). Severe CADRs has a high mortality and there is still no very effective treatment strategy. Methazolamide is a common oral drug to reduce intraocular pressure, which is widely used in glaucoma and reducing intraocular pressure after eye surgery. However, Methazolamide could cause Stevens-Johnson syndrome(SJS) and Toxic epidermal necrolysis(TEN). In our previous studies, we confirmed that Methazolamide induced SJS/TEN was strongly associated with HLA-B*59:01 allele, but the explicit mechanism that Methazolamide interact with HLA-B*59:01 molecule, activate the T cell and lead to severe hypersensitivity were still unknown. Therefore, on the basis of verifying the association between Methazolamide induced SJS/TEN and HLA-B*59:01 allele, we culture the Methazolamide specific T cell in vitro and identify its phenotype and immunological function. By means of cytological and molecular biological experiments, we explore the way that Methazolamide interacts with HLA-B*59:01 molecule, distinguish the specific region of the HLA-B*59:01 molecule and its specific peptides bonded to Methazolamide and verify the results by bioinformation analysis. This research provide the foundation for the primary prevention and precise treatment of Methazolamide induced SJS/TEN, and will be helpful to illuminate immunological and structural biological pathogenesis of SJS/TEN.

药疹是常见的药物不良反应，重症药疹死亡率高，治疗缺乏有效手段。醋甲唑胺为常用口服降眼压药物，广泛用于青光眼治疗及眼科术后降眼压，但醋甲唑胺可致重症药疹SJS/TEN。我们前期实验证实醋甲唑胺所致SJS/TEN与HLA-B*59:01基因强相关，但醋甲唑胺与HLA-B*59:01分子作用激活T细胞，诱发超敏反应导致SJS/TEN发生的具体机制仍不明确。为此，我们在验证醋甲唑胺所致SJS/TEN与HLA-B*59:01基因相关性的基础上，体外培养醋甲唑胺特异性T细胞，明确其表型及功能；通过细胞学和分子生物学实验，探究醋甲唑胺与HLA-B*59:01分子相互作用的方式，识别醋甲唑胺结合HLA-B*59:01分子的关键部位及其结合的特异性自身多肽，并通过生物信息学分析验证。本研究将为醋甲唑胺所致SJS/TEN的一级预防和精准治疗奠定基础，并为SJS/TEN发病机制的探讨提供免疫学和结构生物学证据。

药疹是常见的药物不良反应，重症药疹死亡率高，治疗缺乏有效手段。醋甲唑胺为常用口服降眼压药物，广泛用于青光眼治疗及眼科术后降眼压，但醋甲唑胺可致重症药疹SJS/TEN。我们前期实验证实醋甲唑胺所致SJS/TEN与HLA-B*59:01基因强相关，但醋甲唑胺与HLA-B*59:01分子作用激活T细胞，诱发超敏反应导致SJS/TEN发生的具体机制仍不明确。为此，我们首先对收集的27例醋甲唑胺导致SJS/TEN患者临床特征及预后进行回顾性研究。随后我们提取27例醋甲唑胺导致药物过敏的患者外周血进行HLA测序，并与正常健康人群（使用 10689 健康中国人数据库）进行统计比较，发现 HLA-B*59:01 在 醋甲唑胺所致的 SJS/TEN 组中的携带率为74.07%，在健康人群对照组为 0.36%（Pc= 9.67926E-24，OR= 794.29，95% CI 93.09-6777.08),因此醋甲唑胺所致 SJS/TEN 的 HLA 易感基因定位为 HLA-B*59:01。我们在验证醋甲唑胺所致SJS/TEN与HLA-B*59:01基因相关性的基础上，体外培养醋甲唑胺特异性T细胞明确其表型及功能。进一步利用有限稀释法获得醋甲唑胺特异性T细胞克隆株，发现其主要以CD8+克隆株为主，通过体外培养发现，明确醋甲唑胺药物特异性T细胞克隆株体外增殖具有特异性，同时呈剂量依赖，且乙酰唑胺能够与其发生交叉反应，诱导醋甲唑胺特异性T细胞增殖。且T细胞以HLA I类依赖的方式特异性识别醋甲唑胺药物引起超敏反应。本研究将为醋甲唑胺所致SJS/TEN的一级预防和精准治疗奠定基础并为SJS/TEN发病机制的探讨提供免疫学和结构生物学证据。