Tim-3通过CXCR1/CXCL8轴调控胆管上皮细胞异常增殖的机制研究

The abnormal proliferation of Bile Duct Epithelial Cells is an important pathological change of Primary Biliary Cirrhosis (PBC) while the mechanisms are unclear. Recently, it has been found that CXCR1/CXCL8 axis is associated with abnormal proliferation of Epithelial Cells. However, the underlying regulatory mechanisms are not clear. NK cells are the major source of intrahepatic CXCR1 and CXCL8. The expression of CXCR1 and CXCL8 in NK cells could be regulated by Tim-3 (T cell immunoglobulin and mucin-domain-containing molecule-3) through affecting NK differentiation. Our preliminary studies showed high expression of Tim-3 and Bile Duct Epithelial cells hyperplasia in liver with PBC patients. We speculate that Tim-3 regulate the proliferation of Bile Duct Epithelial Cells through CXCR1/CXCL8 axis. In this research, we will silence and over express Tim-3 in NK cells with RNAi and over-expressed Tim-3 plasmid in our established animal model of PBC. Then, we will investigate whether Tim-3 regulates the proliferation of Bile Duct Epithelial Cells through CXCR1/CXCL8 axis in PBC mice. Moreover, the ERK and NF-κB signal pathways should be evaluated in this condition. 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.

胆管上皮细胞异常增殖是原发性胆汁性肝硬化（PBC）重要的病理改变，机制未完全明了。研究表明上皮细胞异常增殖与CXCR1/CXCL8轴相关，但其调控机制并不清楚。NK细胞是肝内CXCR1和CXCL8的主要来源，Tim-3在NK细胞上转录最多且可调控NK细胞上CXCR1和CXCL8的表达。我们的前期研究发现PBC患者肝内Tim-3高表达、胆管上皮细胞增殖。故推测Tim3通过NK细胞影响CXCR1/CXCL8轴，进而调控胆管上皮细胞增殖。本课题在前期已建立PBC模型的基础上，RNAi沉默Tim-3、构建Tim-3过表达质粒，观察沉默和过表达Tim3后CXCR1、CXCL8表达及对胆管上皮细胞增殖的影响，阐明PBC发病中Tim-3通过调控NK细胞亚群比例改变CXCR1/CXCL8轴，从而通过ERK和NF-κB通路调控胆管上皮细胞增殖的作用及可能机制。为揭示PBC发病机制、寻找新治疗靶点提供依据。

肝内胆管上皮细胞增生是原发性胆汁性胆管炎（PBC）早期的重要病理改变，但其机制尚不明了。结合国内外研究及前期研究成果，我们推测Tim-3通过NK细胞影响CXCR1/CXCL8轴，进而调控胆管上皮细胞增殖。为此，我们从动物、细胞、分子多个层面展开研究。首先，在2OA-BSA与polyI:C联合腹腔免疫建立PBC动物模型基础上，组织病理学观察PBC小鼠肝脏淋巴细胞浸润及胆管上皮细胞不典型性增生情况。检测肝脏、脾脏、外周血NK细胞Tim-3及CXCR1、CXCR3表达，组织病理学和蛋白印迹检测Tim-3、Gal-9、P65、P50、ERK1/2、p-ERK1/2蛋白表达；阻断NK细胞Tim-3通路，观察CXCR1、CXCR3和IFN-γ的表达。阻断CXCR1/CXCL8轴、NF-κB-ERK通路观察肝脏炎症变化、胆管上皮细胞的增殖变化情况。经过以上研究，我们发现胆管上皮增生是早期PBC的病理改变。CXCR1/CXCL8轴参与PBC小鼠肝内炎症的调控，经Reparixin阻断该通路后肝脏炎症明显减轻，肝脏TNF-α、IFN-γ、IL-6 mRNA水平降低。此外，CXCR1/CXCL8轴通过NF-κB及ERK信号通路调控人肝内胆管上皮细胞异常增殖。经PDTC处理后小鼠肝脏内p65、p50表达降低，经PD98059处理后小鼠肝脏内p-ERK表达也降低，提示PDTC和PD98059能够特异性抑制NF-κB和ERK信号通路中的表达。Tim-3/Gal-9在PBC小鼠中表达失调，血清sTim-3较对照组小鼠降低。Tim-3通路可上调NK细胞表达CXCR1和分泌IFN-γ，抑制CXCR3免疫调控作用，对NK细胞趋化功能具有潜在调控作用。综合以上结果，PBC时Tim-3/Gal-9轴功能紊乱，能调控NK细胞CXCR1、CXCR3的表达参与PBC发病。此外，血清sTim-3水平变化也与PBC疾病相关。CXCR1/CXCL8 轴和NF-κB-ERK信号通路在PBC疾病的发生发展中发挥重要作用。本项目揭示了PBC时Tim-3/Gal-9 功能紊乱，并能通过CXCR1/CXCL8 轴调控肝脏局部免疫微环境，促进胆管上皮细胞增殖。为今后临床研究和开发Tim-3的潜在应用提供理论基础，尤其血清sTim-3水平在临床预后评估中的重要价值。