RORγtN277D突变对TH17细胞的调控机制及其在自身免疫性肝炎中的作用

Autoimmune hepatitis, formerly called lupoid hepatitis, is a chronic, autoimmune disease of the liver that occurs when the body's immune system attacks liver cells causing the liver to be inflamed as a result of failure of immune tolerance mechanisms in a genetically susceptible host. The interpretation of genetic susceptibility is not clear yet. We sequenced RORC gene of peripheral blood mononuclear cell collected from AIH patients and healthy controls, which encodes TH17 master transcription factor RORγt. N277D mutation (designated as RORM here after) was identified from 10 out of 48 patients. RORM is more potent to transactivate downstream genes in reporter assay and drive TH17 differentiation in vitro differentiation assay, in comparison with wide type counterpart. Therefore, our hypothesis is RORγt N277D facilitates TH17 differentiation and function in liver result in the failure of immune tolerance and contributes to genetic susceptibility. To verify increased activity of RORM, primary T lymphocytes will be transduced to express wt or mutated RORγt and tested by IL17 production and ability to induce experimental autoimmune hepatitis (EAH) in Rag1-/- mice. To further demonstrate the function of this mutant, RORM mice will be established and subjected to EAH. This study will partially demonstrate the molecular mechanism for genetic susceptibility of autoimmune hepatitis and contribute to the development of screening assay.

遗传易感性是自身免疫性肝炎（AIH）的主要病因，但遗传易感性的本质及其致病机理仍未阐明。本课题组前期在48例AIH患者中发现10例患者TH17特征转录因子RORγt基因277位天冬酰胺突变为天门冬氨酸（下文以ROR-M表示该突变基因型）。在报告基因系统中，体外过表达突变的RORγt较野生型有更高的转录活性；在TH17体外分化实验中，ROR-M显著促进TH17分化及IL17合成。我们推测，该突变上调RORγt转录活性，增强肝组织中TH17细胞分化及功能，因而携带ROR-M的人群易于罹患AIH。我们拟在前期基础上，利用TH17体外分化模型、实验性AIH小鼠模型，采用逆转录病毒转导原代T淋巴细胞、构建ROR-M敲变小鼠，观察该突变对RORγt活性及TH17功能的影响，以及该突变能否增加小鼠对实验性自身免疫性肝炎的易感性。本研究有助于部分阐明AIH遗传易感性的可能分子基础，为早期筛查提供理论依据。

遗传易感性是自身免疫性肝炎（AIH）的主要病因，本课题组发现AIH患者中有较高比例患者的TH17特征转录因子RORγt基因277位天冬酰胺突变为天门冬氨酸（下文以RORM表示该突变基因型）。在报告基因系统中，体外过表达突变的RORγt较野生型有更高的转录活性；在TH17体外分化实验中，RORM显著促进TH17分化及IL17合成。我们的工作发现该突变上调RORγt转录活性，增强肝组织中TH17细胞分化及功能，因而携带RORM的人群易于罹患AIH。我们利用TH17体外分化模型、实验性AIH小鼠模型，采用逆转录病毒转导原代T淋巴细胞、构建RORM敲变小鼠，观察到该突变对RORγt活性及TH17功能的影响，且该突变可能增加小鼠对实验性自身免疫性肝炎（EAH）的易感性。明确了自身免疫性肝炎的关键生物学过程以及免疫细胞浸润情况，并发现MB能缓解EAH的肝损伤。本研究有助于部分阐明AIH遗传易感性的可能分子基础，为早期筛查提供理论依据。