原发性胆汁性肝硬化时单核细胞调控NK细胞功能的机制研究

Primary biliary cirrhosis (PBC) is an autoimmune disease, and the intrahepatic bile duct epithelial cells are the target cells of the immune attack. Recently, it has been found that the activated natural killer (NK) cells destroy biliary epithelial cells directly, and monocytes regulate the activation of NK cells. However, the underlying specific activation and the regulatory mechanisms are not clear. Our preliminary study showed that in the in liver of PBC patients, there was an increased NK cell activity, which was accompanied by increased expression of major immunogen complex (MIC) and soluble cytokines in monocytes. We speculate that monocytes may regulate the functions of NK cells through two pathways, interaction between MIC expressed on monocytes and a surface C-type lectin-like activating receptor, NKG2D, on NK cells, and soluble cytokines expressed by monocytes. In this research, we will establish an animal model of PBC by intraperitoneal injection of 2 - methyl octanoate (2OA) and Poly I: C in C57BL/6 female mice. Then, Kupffer cells (monocytes in the liver), NK cells, and biliary epithelial cells in the liver were separated and used to investigate whether monocytes regulate the functions of NK cells through MIC-NKG2D interaction and soluble cytokines (including IL-10, IL-12, IL-15, IL-18 and IL-23) in PBC mice. Moreover, the peripheral blood and liver biopsies of patients with PBC will be used to verify the results in the animal model. Our project will provide novel insights into the pathogenesis of PBC, which may serve as a new theoretical basis for further identification of new therapeutic targets.

原发性胆汁性肝硬化（PBC）是一种自身免疫性疾病，肝内胆管上皮细胞是免疫攻击的靶细胞。最近发现活化的NK细胞可直接破坏胆管上皮细胞，而单核细胞对NK细胞活化有调控作用，但具体调控机制尚不明确。我们前期发现PBC患者NK细胞活性增强，单核细胞高表达MIC以及可溶性细胞因子，推测单核细胞通过MIC与NK细胞表面活化性受体NKG2D相互作用和可溶性细胞因子两种方式调控NK细胞功能。本课题拟采用2-辛酸甲酯（2OA）及Poly I：C腹腔注射C57BL/6雌性小鼠建立PBC动物模型，分离肝内Kupffer细胞、NK细胞并研究其相互作用，阐明PBC小鼠单核细胞通过细胞接触的MIC-NKG2D相互作用和可溶性细胞因子（IL-10、L-12、IL-15、IL-18和IL-23）作用两种方式调控NK细胞功能的机制，并取PBC患者外周血及肝穿组织以验证。将为阐明PBC发病机制、寻找新的治疗靶点提供理论依据。

【研究背景】原发性胆汁性肝硬化发病机制尚不明确。我们的前期研究发现，PBC 患者肝内NK 细胞功能增强，但NK细胞活化机制仍不清楚。【研究内容】应用2OA-BSA与poly I:C联合腹腔免疫建立PBC动物模型，研究PBC小鼠模型肝脏和外周血细胞因子IL-10、IL-12、IL-15、IL-18、TNF-α和IFN-γ表达水平及Kupffer细胞和NK细胞活化性受体表达变化，探讨Kupffer细胞与PBC疾病发生发展的关系。分离小鼠肝内Kupffer细胞、NK细胞进行原代培养，探讨PBC小鼠Kupffer细胞通调控NK细胞功能的机制，以及NKG2D/MIC通路在PBC 发病中的作用，并进行临床验证。为阐明PBC 发病机制提供新的理论依据。【研究结果】①PBC动物模型成功建立②免疫组织化学染色显示模型组肝脏F4/80、RAE-1及NKG2D较对照组增高。流式细胞术显示，NKG2D在外周血表达较低，而在原代肝NK细胞上表达增高；RAE-1及F4/80在外周血及原代Kupffer细胞上表达均增高③Kupffer细胞与NK 细胞共培养，NK细胞表面NKG2D表达增加；Tranwell小室阻断Kupffer细胞与NK 细胞接触，NK 细胞受体表达量下降④IL-12、TNF-α、IFN-γ在PBC小鼠外周血均有分泌，其中IL-12高达8265.14pg/mL；肝内TNF-α主要由Kupffer细胞表达，NK细胞主要分泌IFN-γ；在各培养体系均有分泌，尤其是LPS的刺激下，分泌明显增多；而IL-10、IL-15及IL-18在体内和体外的分泌量较低⑤NK细胞与LPS刺激后的Kupffer细胞共培养后，细胞杀伤活性增强，但与LPS的剂量相关性不明显⑥PBC患者中，随着炎症活动度的加重，NK细胞活化性受体NKG2D的表达上调；而NKG2D的表达随着纤维化程度的加重下降，IL-15表达上调。【结论】2OA-BSA与poly I:C 联合腹腔免疫可成功建立PBC 动物模型。PBC时肝脏内Kupffer细胞、NK细胞增生，且Kupffer细胞能够通过细胞因子及NKG2D/MIC通路激活NK细胞，参与PBC的发生发展。

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