猪急性肝衰竭模型的建立及猪纤维介素的表达

目的 建立一个模拟人类疾病进程的猪急性肝衰竭(ALF)模型,用于ALF治疗药物的临床前安全性评价及疗效评价,检测模型中猪纤维介素(pfg12)的表达情况,为针对fg12的基因治疗提供基础和依据. 方法造模组耳静脉快速注射D-氨基半乳糖盐酸盐,剂量1.2 g/kg;对照组耳静脉快速注射5%的葡萄糖,剂量12 ml/kg.观察两组动物临床表现,肝功能指标和肝组织病理学改变,实时定量PCR检测肝组织中pfg12 mRNA表达,免疫组织化学检测肝组织中pfg12蛋白的表达.采用重复测量数据的方差分析和独立样本t检验进行统计学处理.结果 成功建立了与人在临床表现、肝脏生物化学指标、组织病理学改变相似的猪ALF模型;实时定量PCR检测结果显示造模组猪肝组织中pfg12的mRNA表达水平显著增加,与对照组比较,差异具有统计学意义(t=7.695,P＜0.05).免疫组织化学显示造模组猪肝组织中有明显的pfg12蛋白的表达,主要分布在肝细胞坏死区域的肝细胞、炎症浸润细胞、肝血窦内皮细胞及血管内皮细胞,对照组动物肝组织未见pfg12阳性着色.结论 以D-氨基半乳糖盐酸盐诱导的猪ALF模型可用于评价肝衰竭治疗药物的临床前疗效及安全性;pfg12在猪ALF动物模型的肝组织中异常高表达,提示其参与了ALF时肝细胞坏死的发生和发展过程.

Establishment of pig acute liver failure model and the role of pig fibrinogen-like protein 2

Objective To establish a pig model of fulminant hepatic failure for evaluating the preclinical efficacy of drug treatment on severe hepatitis,and to detect the expression of fibrinogen-like protein2(fg12)prothrombinase in the model,so as to provide basis for gene therapy targeting to fg12 for fulminant hepatic failure.Method D-galactosamine hydrochloride was used to induce pig model of fulminant hepatic failure,and the experiment animals were divided into model group(rapid injection of D-galactosamine hydrochloride by ear vein,a dose of 1.2 g/kg)and negative control group(5%Glucose).Clinical,biochemical and pathological changes of animals were observed.The expression of pigs fg12(pfg12)mRNA in liver tissue was detected by real time RT-PCR,the expression of pfg12 protein in liver tissue was detected by immunohistochemistry.Results A pig model of fulminant hepatic failure WaS successfully established using the D-galactose hydrochloride;Real time RT-PCR of liver fg12 mRNA showed that fg12 mRNA expression was increased significantly in liver tissue of fulminant hepatic failure pig model compared with the control group(P=0.0 1 6);Immunohistochemical staining showed that there were fg12 protein expression in liver tissue of fulminant hepatic failure pig model,mainly in the membrane and cytoplasm of hepatocytes,inflammatory cells,liver sinusoidal endothelial cells and vascular endothelial cells of liver cell necrosis region.However,there are no fg12 positive staining on negative control. Conclusions The pig model of fulminant hepatic failure induced by D-galactosamine hydrochloride is similar to human pathological process and can be used to evaluate the pre-clinical efficacy and safety of drug treatment on fulminant hepatic failure. Abnormal expression of pfg12 at both mRNA level and protein level in the liver of fuiminant hepatic failure pig model shows that pfg12 induced coagulation pathway is also involved in the development of fulminant hepatic failure. Gene therapy targeting fg12 genes for fulminant hepatic failure may provide a new means for the treatment of fulminant hepatic failure.