Mer酪氨酸激酶通路在原发性胆汁性肝硬化巨噬细胞极化中的调控机制研究

Primary biliary cirrhosis is an autoimmune disease characterized by the destruction of intrahepatic bile ducts.The mechanism of IBEC-specific damage by surrounding immune cells in PBC has remained unknown. We have found in the previous study, that macrophages were polarized toward M1 phenotype and the expression of MerTK which can regulate macrophage phagocytosis was significantly decreased at early stage of PBC.The opposite was the case at advanced stage. Therefore, we hypothesized that at early stage of PBC,apoptotic IBEC could not be cleared timely owing to M1 polarization of macrophages induced by defective MerTK, resulting in specific damage of IBEC. Otherwise, macrophages were indcued toward M2 polarization by overexpression of MerTK at advanced stage to participate in fibrosis and cirrhosis of PBC.To confirm this hypothesis, various techniques such as immunohistochemisty, gene overexpression and silence, immunofluorescence and immunoblotting.etc will be used to explore the detailed molecular mechanisms at various levels including clinical, cell,molecular and animal. It will help to explore the mechanism of IBEC-specific damage in PBC and search for the key target, with which macrophages polarization can be switched to realize the goal of dynamic treatment for PBC to some extent.

原发性胆汁性肝硬化（PBC）是以肝内胆管上皮细胞损伤为主要特征的慢性胆汁淤积性自身免疫性肝病。胆管周围浸润的免疫细胞造成胆管特异性损伤是其主要病理特征，但具体机制未明。我们在前期研究发现PBC不同阶段巨噬细胞表型呈动态变化，早期以M1巨噬细胞为主，参与调控作用的MerTK表达下调；晚期以M2为主，MerTK表达上调。因此，我们提出PBC早期MerTK表达缺陷，诱导巨噬细胞向M1极化，无法及时清除凋亡的胆管上皮细胞，导致胆管特异性炎症损伤；而PBC中晚期，MerTK高表达则诱导巨噬细胞M2极化，通过分泌细胞因子参与PBC纤维化、硬化的假说，并拟应用免疫组化、基因过表达及沉默、免疫荧光、免疫印迹等方法，从临床、细胞、分子和动物水平及正反两方面验证这一假说，阐明其具体分子机制，对于寻找PBC关键靶点，扭转不同阶段巨噬细胞极化方向具有重要意义，为个体化诊疗PBC提供思路。

原发性胆汁性肝硬化（PBC）是以肝内中小胆管上皮细胞损伤为主要特征的慢性胆汁淤积性自身免疫性肝病，病因和发病机制未明。经本课题研究，取得以下结果：一、酪氨酸激酶家族受体和配体在PBC患者外周血单个核细胞存在异常表达。PBC患者血清可溶型sMer和sTyro-3水平显著高于健康对照组（P<0.05）；sMer和sTyro-3与AKP,GGT,TBA呈显著正相关。流式结果显示早期PBC患者M1巨噬细胞比例显著高于晚期患者（P<0.05）；而M2型巨噬细胞在晚期PBC患者显著高于早期患者（P<0.05）。早期PBC患者膜型Mer水平显著低于晚期患者（P<0.01）。M2/M1比值与膜型Mer，胆红素和碱性磷酸酶呈显著正相关（r=0.57,r=0.81,r=0.71,P均<0.01）。二、Mer受体通过调控p-Akt信号通路，影响M2巨噬细胞诱导因子IL-10和IL-13表达，促进M2巨噬细胞极化。三、PolyI:C注射C57BL/6雌性小鼠16周可见PBC样炎症病变，免疫荧光检测肝组织胆管细胞PDC-E2表达增高。清除再转输过表达MerTK的BMDMs后PolyI:C注射组肝细胞分布排列较规则，少量炎症细胞浸润。清除再转输基因沉默MerTK的BMDMs后PolyI:C注射组，可见汇管区有明显炎性细胞浸润。清除再转输过表达MerTK的BMDMs后PolyI:C注射组MerTK，Arg1、MRC1、FIZZ1和CHI3L3水平高于清除再转输基因沉默MerTK的BMDMs后PolyI:C注射组，而CD8+CD44+T细胞和IL-12水平降低。此外，PBC患者趋化因子SDF-1，CCL11、CCL24和CCL26表达显著增高，提示可能与Th17细胞和嗜酸性粒细胞的趋化有关。因此，通过临床、细胞和动物研究，我们发现PBC早期MerTK表达缺陷，诱导巨噬细胞向M1极化，无法及时清除凋亡的胆管上皮细胞，导致胆管特异性炎症损伤；而PBC中晚期，MerTK高表达则诱导巨噬细胞M2极化,参与PBC纤维化。