急性布加综合征小鼠模型的建立及其肝脾肾组织中蛋白酶激活受体-1的表达

Establishment of mice model of acute Budd-Chiari syndrome and expression of protease-activated receptor-1 in liver,spleen and kidney tissues

目的建立急性布加综合征(Budd-Chiari syndrome,BCS)小鼠模型,并初步探讨蛋白酶激活受体-1(protease activated receptor-1,PAR-1)在小鼠模型肝脾肾组织中的表达。方法采用部分结扎肝上段下腔静脉法建立小鼠急性BCS模型,观察小鼠术后一般情况,血清指标及病理改变;采用免疫组化及RT-PCR法检测小鼠肝脾肾组织中PAR-1的蛋白和m RNA表达水平。结果实验组小鼠相继出现不同程度的食欲差、活动少、毛发发黄、腹水等症状;解剖后肝脏、脾脏、肾脏可见体积增大、色暗红的淤血表现。建模2周后,实验组小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TIBL)及直接胆红素(DBIL)较对照组均升高,差异有统计学意义(t=7. 484、6. 258、7. 634、6. 874,P=0. 000、0. 000、0. 000);与对照组相比,实验组小鼠2周后血清肌酐(CREA)升高,差异有统计学意义(t=2. 863,P=0. 012),血清尿素氮(BUN)水平虽有所上升,但差异无统计学意义(t=1. 313,P=0. 209);红细胞(RBC)、白细胞(WBC)及血小板(PLT)两组比较差异均无统计学意义(t=0. 224、0. 758、1. 210,P=0. 826、0. 460、0. 245)。建模2周后,实验组小鼠镜下示肝小叶中央静脉及肝窦扩张,肝索排列紊乱,部分小叶中央静脉周围出现斑片状坏死及炎性细胞浸润;部分脾细胞出现变性、坏死,脾脏白髓和红髓界限不清;肾小管上皮细胞水肿,肾小静脉扩张。免疫组化结果显示PAR-1主要在肝脏组织中的血管内皮细胞、肝窦内皮细胞和肾脏组织中的肾小管上皮细胞表达,而在脾脏组织中呈弱表达,偶见于巨噬细胞;实验组小鼠肝组织中PAR-1蛋白及mRNA的表达水平明显高于对照组(P蛋白=0. 003,PmRNA=0. 000),而在脾脏和肾脏组织中差异无统计学意义(脾脏:P蛋白=0. 617,PmRNA=0. 194;肾脏:P蛋白=0. 260,PmRNA=0. 496)。结论通过部分结扎肝上段下腔静脉法模拟急性BCS模型是可行、有效的,PAR-1很可能参与了BCS腹腔脏器淤血损伤的病理生理过程,特别是在淤血性肝损伤中发挥重要作用。

Objective To establish mice model of acute Budd-Chiari syndrome(BCS),and to preliminarily investigate the expression of protease activated receptor-1(PAR-1)in liver,spleen and kidney tissues of mice.Methods The acute BCS model of mice was established by partial ligation of the supra-hepatic inferior vena cava.The general condition,serum index and pathological changes of the mice were observed.Immunohistochemistry and RT-PCR were performed to detect the PAR-1 protein and m RNA expression levels in mice liver,spleen and kidney tissues.Results The mice in the experimental group showed different degrees of poor appetite,less activity,yellow hair and ascites.After dissection,the liver,spleen,and kidney showed congestion performance such as an increase in volume and dark red color.After 2 weeks of modeling,the serum levels of ALT,AST,TBIL and DBIL in the experimental group were higher than those in the control group,with significant difference(t=7.484,6.258,7.634,6.874,P=0.000,0.000,0.000).Compared with the control group,serum creatinine(CREA)in the experimental group increased 2 weeks later,with statistically significant difference(t=2.863,P=0.012),although the serum urea nitrogen(BUN)level increased,the difference was not statistically significant(t=1.313,P=0.209).There were no significant differences between the two groups in RBC,WBC and PLT(t=0.224,0.758,1.210,P=0.826,0.460,0.245).Two weeks after modeling,the mice in the experimental group showed the expansion of central hepatic vein and hepatic sinus under microscope,disordered hepatic cords,patchy necrosis and inflammatory cell infiltration appeared around the central vein in some of the lobules.Some spleen cells showed degeneration and necrosis,and the spleen white pulp and red pulp were unclear;the renal tubular epithelial cells were edematous and the renal venules were dilated.Immunohistochemistry showed that PAR-1 was mainly expressed in vascular endothelial cells,sinusoidal endothelial cells in liver tissues and renal tubular epithelial cells in kidney tissue,but it was weakly expressed in spleen tissues,occasionally in macrophages.The expression levels of PAR-1 protein and m RNA in the liver tissues of mice in the experimental group were significantly higher than those in the control group(Pprotein=0.003,PmRNA=0.000),but there was no significant difference between the two groups in spleen and kidney tissues(spleen:Pprotein=0.617,PmRNA=0.194;kidney:Pprotein=0.260,PmRNA=0.496).Conclusion It is feasible and effective to simulate the acute BCS model by partial ligation of the supra-hepatic inferior vena cava.PAR-1 is likely to participate in the pathophysiological process of BCS abdominal organ congestion injury,especially playing an important role in the congestive liver injury.