原发性胆汁性胆管炎小鼠模型肝内趋化因子CXCL13的表达

Expression of intrahepatic chemokine CXCL13 in a mouse model of primary biliary cholangitis

目的原发性胆汁性胆管炎(PBC)患者肝内趋化因子CXCL13表达显著增加,但其来源和机制尚不明确。文章旨在通过建立PBC小鼠模型,探索PBC小鼠肝内趋化因子CXCL13的表达及其主要来源细胞。方法采用随机数字表法将C57BL/6小鼠分为实验组[n=20,腹腔注射聚肌苷酸-聚胞苷酸(Poly I:C,2次/周,共12周)]和对照组(n=10,腹腔注射,系同实验组等体积磷酸盐缓冲液)。酶联免疫吸附试验(ELISA)测定血清AMA水平,HE染色观察肝组织炎症细胞浸润情况。原位灌注酶消化结合磁珠分选法分离小鼠肝内Kupffer细胞、肝血窦内皮细胞以及肝内淋巴细胞,荧光定量PCR检测肝组织以及上述细胞亚群CXCL13 mRNA表达水平。结果实验组小鼠血清AMA水平随建模时间延长逐渐上升,首次注射Poly I:C后第4、8、12周阳性率分别为5.9%、52.9%以及76.5%,而对照组小鼠血清AMA水平在整个建模过程中均呈现较低水平,仅在第12周时有2只小鼠AMA水平略高于阳性界值。实验组和对照组小鼠肝组织HE染色结果表明,实验组小鼠肝内汇管区大量炎症细胞浸润,而对照组小鼠肝内未见炎症细胞浸润。流式细胞术检测实验组小鼠Kupffer细胞、肝血窦内皮细胞的分离纯度分别为76%~80%、68%~72%。与Kupffer细胞CXCL13 mRNA表达[2.34(0.22-8.64)]比较,肝血窦内皮细胞、肝内淋巴细胞表达下降[0.27(0.03-1.64)、0.05(0-0.22),P<0.05]。结论 Poly I:C诱导建立的PBC小鼠模型中肝内升高的趋化因子CXCL13主要来源于Kupffer细胞。

mental group were intraperitoneally injected with polyriboinosinic polyribocytidylic acid （Poly I：C ） while the mice in control group were injected with PBS of the same volume. The level of serum AMA was quantified by ELISA and intrahepatic inflammatory cells were assessed by HE staining. Kupffer cells, liver sinusoidal endothelial cells, and infiltrating lymphocytes in the liver of mice were collected by in situ perfusion enzyme digestion and magnetic bead separation methods. The transcriptional level of intrahepatic CXCL13 in liver tissues and cell subpopulations were detected by qPCR. Results The serum AMA titers of the mice in experiment group increased gradually with the prolonging of modeling time and the positive rates at the 4th, 8th, and 12th week after the first injection of Poly I： C were 5.9%, 52.9% and 76.5% respectively. While the serum AMA titers of the mice in control group were at a lower level through the modeling process, with only 2 mice presenting a little higher level above positive cutoff value at the 12th week. The results of HE staining in liver tissues of both groups showed that there were a great amount of intensely infiltrating inflammatory cells in the mice of experimental group while no inflammatory cell infiltration were found in the mice of control group. The separation purity of Kupffer cells and liver sinusoidal endothelial cells in the mice of experiment group tested by flow cytometry were 76%-80%, 68%-72% respective- ly. Compared with the CXCL13 mRNA level in Kupffer cells [ 2.34（0.22-8.64） ], the expression levels in liver sinusoidal endothelial cells and infiltrating lymphocytes declined[0.27（0.03-1.64）, 0.05（0-0.22）, P〈0.05]. Conclusion The ehemokine CXCL13 is predominantly produced by Kupffer ceils in the liver of PBC mice established by Poly I ： C injection.