LAMP2及其截断蛋白参与原发性胆汁性肝硬化胆汁排泄障碍的机制研究

Primary biliary cirrhosis (PBC) is characterized by the chronic intrahepatic cholestasis, which is related to the abnormality of hepatocyte canalicular surfaces. In our previous study, hepatocyte canalicular membrane fraction isolated from the liver of PBC patients was used as immunogens to produce hybridomas and mAb2 (monoclonal antibody 2) was obtained which could contribute to pathological diagnosis in PBC patients. In normal human liver, mAb2 antigen exclusively localized to the membrane of endosomes around hepatocyte canalicular membrane. However, mAb2 antigen in the liver of PBC patients expressed much highly and the polarized distribution disappeared, localizing to the perinuclear lysosomes. Meanwhile, there was the soluble form of mAb2 antigen in the cytoplasm. With a series of assays, it could be confirmed that the antigen recognized by mAb2 is LAMP2. As described previously, LAMP2 was involved in regulating the hepatocyte transport systems, which suggested that abnormality of LAMP2 might be responsible for the cholestasis in PBC patients. Therefore, the present study aims to further identify the characterization of the soluble form of LAMP2 in the hepatocyte cytoplasm of PBC patients, and clarify potential roles and mechanisms of LAMP2 and their soluble form of LAMP2 involved in regulating bile excretion of hepatocyte canalicular membrane, which may help illuminate the mechanisms of intrahepatic cholestasis and find the novel therapeutic targets in PBC patients.

肝内胆汁淤积是PBC的重要病理改变，肝细胞胆管侧细胞膜异常与其发病密切相关。抗体mAb2是本课题组以PBC患者肝细胞胆管侧膜性组分为免疫原，筛选获得的对PBC病理诊断具有重要价值的特异性单抗,其在正常肝脏中呈极性分布，位于肝细胞胆管侧细胞膜周围的内体囊壁上；而在PBC患者肝组织中，其表达明显增高且分布紊乱，位于细胞核周围的溶酶体上，胞浆中伴有可溶性分子的存在；实验证实mAb2所识别的抗原为LAMP2。既往发现LAMP2对细胞内运输具有重要的调控功能，提示其可能参与肝细胞胆管侧细胞膜对胆汁排泄的调控，其异常可导致肝内胆汁淤积。因此，本课题拟在前期研究基础上，明确PBC患者肝细胞中可溶性LAMP2的截短形式；探讨LAMP2及其截断蛋白对肝细胞胆管侧细胞膜胆汁排泄功能的影响、调控机制及其相互作用蛋白，为阐明LAMP2参与PBC胆汁排泄障碍的分子机制，寻找PBC新的药物靶点提供理论依据和实验基础。

LAMP-2分子在多种生物学进程中发挥重要作用，但其在胆汁排泄中的作用仍知之甚少。本研究的目的在于探讨LAMP-2在胆汁淤积性疾病中的作用，以及LAMP-2参与肝细胞胆汁排泄的可能机制。我们在前期成功鉴定1F9抗原为LAMP-2，并且原发性胆汁性肝硬化（PBC）患者肝脏组织LAMP-2表达增强、分布紊乱的基础上，发现：1. PBC组织中LAMP-2 mRNA表达增强；2. PBC患者外周血LAMP-2水平明显升高；3. 借助转录激活因子样效应核酸酶技术（TALENs），成功构建了LAMP-2基因敲除大鼠模型，惊喜发现该模型大鼠肝脏表现有胆汁淤积的现象，体内研究发现LAMP-2通过调控Mrp2在肝细胞毛细胆管膜上的定位，影响肝细胞胆汁排泄过程。该结果进一步得到了体外细胞学实验的支持。结论：LAMP-2分子与肝细胞胆汁排泄密切相关，其可以通过调控肝细胞毛细胆管膜胆汁排泄分子发挥作用。