【摘要】 目的 探讨泡球蚴蛋白对肝星状细胞中纤维化关键基因二肽基肽酶-4（Dipeptidyl Peptidase 4,DPP4）表达的调控机制。方法 免疫组织化学法检测二肽基肽酶-4在泡型棘球蚴病患者肝脏病灶近端与远端的表达情况。体外培养小鼠肝星状细胞系JS1,以60μg/mL浓度的泡球蚴蛋白（Echinococcus multilocularis Protein, EmP）刺激JS1细胞，检测细胞活化及Wnt通路的激活情况，Wnt通路抑制剂（IWP-2）和激动剂（LY2090314）靶向干预检测肝星状细胞活化标志物COL1A1、α-SMA与PPARγ,Wnt通路关键分子Wnt5a、β-catenin、GSK3β以及DPP4的表达水平。结果 免疫组化检测结果显示泡型棘球蚴病患者肝脏病灶近端DPP4的表达（5927±987.1）显著高于病灶远端（2478±696.9）,差异有统计学意义（t=9.026,P<0.05）;EmP可显著促进JS1细胞活化并促进DPP4的表达，差异有统计学意义(F_DPP4=10.65,P<0.05);与对照组相比，EmP刺激组Wnt通路激活，Wnt5a、β-catenin、p-GSK3β的表达水平上升，差异有统计学意义(WB:F_Wnt5a=18.28,F_β-Catenin=14.98,F_P-GSK3β=28.52;RT-qPCR:F_Wnt5a=27.29,F_CTNNB1=21.24,F_GSK3β=7.974,P<0.05);Wnt通路抑制剂IWP-2干预可抑制EmP蛋白对α-SMA、DPP4的表达的促进作用，上调PPARγ的表达，差异有统计学意义(WB:F_DPP4=26.27,F_α-SMA=126.4,F_PPAR-γ=8.187;RT-qPCR:F_DPP4=41.23,F_ACTA2=185.2,F_PPARG=136.2,P<0.05);与EmP刺激组相比，Wnt通路激动剂可促进JS1细胞的活化及DPP4的表达，差异有统计学意义(WB:F_DPP4=151.0;RT-qPCR:F_DPP4=29.71,P<0.05)。结论 EmP蛋白可通过激活Wnt/β-Catenin通路促进纤维化关键基因DPP4的表达。更多还原

【Abstract】 Objective To determine the regulatory mechanism of Echinococcus multilocularis protein on the expression of Dipeptidyl Peptidase 4（DPP4）,a key gene for fibrosis in hepatic stellate cells. Methods Mouse hepatic stellate cell line JS1 was cultured in vitro,and JS1 cells were stimulated with E. multilocularis protein（EmP）concentration of 60 μg/mL to detect cell activation and activation of Wnt pathway, the expression of Dipeptidyl Peptidase4 in the closed and distant liver lesions of alveolar echinococcosis patients was detected by immunohistochemistry.Wnt pathway was targeted by Wnt pathway inhibitor（IWP-2） and agonist（LY2090314）,and the expression levels of hepatic stellate cell activation markers COL1A1,α-SMA and PPARγ,Wnt pathway key molecules Wnt5a, β-catenin, GSK3β and DPP4 were detected Results EmP could significantly promote the activation of JS1 cells and the expression of DPP4,with statistical significance(F_DPP4=10.65,P<0.05),The expression of DPP4 in the closer to the liver lesions（5927±987.1）in alveolar echinococcosis patients was significantly higher than that in the distal end of liver lesions, with statistical significance（2478±696.9）,(t=9.026,P<0.05 Compared with the control group, the Wnt pathway was activated in EmP stimulation group, and the expression levels of Wnt5a, β-catenin and P-GSK3β were increased, with statistical significance(WB:F_Wnt5a=18.28,F_β-Catenin=14.98,F_P-GSK3β=28.52;RT-qPCR:F_Wnt5a=27.29,F_CTNNB1=21.24,F_GSK3β=7.974,P<0.05). Wnt pathway inhibitor IWP-2 could inhibit the promoting effect of EmP protein on the expression of α-SMA and DPP4,and up-regulate the expression of PPARγ,with statistical significance(WB:F _DPP4 =26.27,F_α-SMA =126.4,F_PPAR-γ=8.187;RT-qPCR:F_DPP4 =41.23, F_ACTA2=185.2,F_PPARG=136.2,P<0.05).Compared with EmP stimulation group, Wnt pathway agonists could further promote JS1 cell activation and DPP4 expression, and the difference was statistically significant(WB:F_DPP4=151.0;RT-qPCR :F_DPP4=29.71,P<0.05). Conclusion EmP can promote the expression of DPP4,a key gene for fibrosis, by activating the Wnt/β-Catenin pathway.更多还原