小鼠肝多房棘球蚴感染模型中细胞焦亡NLRP3/Caspase-1通路的初步研究  

Preliminary research on pyroptosis NLRP3/Caspase-1 pathway in mouse model of hepatic alveolar echinococcosis infection

作　　者：乜茹 刘川川 李文登 庞明泉[1,2] 尹凤娇 冶赓博 王志鑫 樊海宁[1,2] NIE Ru;LIU Chuanchuan;LI Wendeng;PANG Mingquan;YIN Fengjiao;YE Gengbo;WANG Zhixin;FAN Haining(Department of Hepatopancreatobiliary Surgery,Affiliated Hospital of Qinghai University,Xining 810001,P.R.China;Qinghai Research Key Laboratory for Echinococcosis,Xining 810001,P.R.China;Graduate School of Qinghai University,Xining 810016,P.R.China)

机构地区：[1]青海大学附属医院肝胆胰外科,西宁810001 [2]青海省包虫病研究重点实验室,西宁810001 [3]青海大学研究生院,西宁810016

出　　处：《中国普外基础与临床杂志》2023年第6期666-673,共8页Chinese Journal of Bases and Clinics In General Surgery

基　　金：青海省科技厅项目(项目编号:2019-SF-131);青海大学附属医院中青年科研基金项目(ASRF-2019-YB-04)。

摘　　要：目的观察小鼠肝多房棘球绦虫(Echinococcus multilocularis,Em)感染模型中细胞焦亡NLRP3/Caspase-1通路中关键蛋白的表达并探讨其相关性。方法选取25只BALB/c小鼠按随机数字表分为对照组和感染组,感染组以0.2 mL原头节悬液(含原头节3000个)注射于肝脏被膜下,建立肝Em感染模型,对照组不予处理,常规饲养。分别于模型建模后1、2、3及5个月处死小鼠,摘取肝脏,观察肝脏大体形态,行HE染色、透射电镜观察组织病理学改变;行免疫组化和免疫印迹法检测肝脏组织中细胞焦亡半胱氨酸天冬氨酸蛋白酶-1(cysteinyl aspartate specific proteinase-1,Caspase-1)/Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)通路中关键蛋白的表达,采用ELISA测定细胞焦亡下游因子IL-1β的表达水平。结果与对照组相比,感染组小鼠肝组织表面囊性病灶随时间延长逐渐增大并凸出于肝脏表面;HE染色可见肝脏病灶出现不同程度的炎性细胞浸润、纤维增生等多种病理改变。Em感染2个月组透射电镜观察可看到肝细胞的细胞膜断裂、不连续,符合细胞焦亡的“打孔”现象。ELISA检测结果显示Em感染1、2、3和5个月后小鼠肝脏组织匀浆中IL-1β浓度均高于对照组的浓度,差异有统计学意义(F=127.2,P<0.05)。免疫组化检测结果显示,Caspase-1和NLRP3在Em感染1、2、3及5个月的小鼠肝脏中阳性细胞比率均高于对照组,差异有统计学意义(F=114.6,P<0.05;F=85.89,P<0.05)。蛋白免疫印迹结果显示,Caspase-1、消皮素D(gasdermin D,GSDMD)和NLRP3蛋白在感染组小鼠肝脏中的相对表达水平呈现先升高(其中Caspase-1和GSDMD的表达于感染1个月时升至最高,NLRP3的表达于感染2个月时升至最高),后降低的趋势,各时相感染组与对照组比较以及各时相感染组之间比较差异均有统计学意义(均P<0.05)。结论采用“切开皮肤经腹壁肌层肝穿刺法”建立小鼠Em感染模型是可行的;小鼠肝Em感染后Objectives To observe the expression of key proteins in the NLRP3/Caspase-1 pathway of pyroptosis in the mouse model of hepatic Echinococcus multilocularis(Em)infection and explore its correlation.Methods Twentyfive BALB/c mice were randomly divided into the control group and the infected group.The infected group was injected with 0.2 mL suspension of protoscolex(including 3000 protoscoleces)injected under the liver capsule to establish a model of secondary infection with hepatic Em.The control group was treated without any treatments and conventional feeding was conducted.The mice were sacrificed at 1,2,3,and 5 months after infection.The liver was harvested and observed for gross morphology.HE staining and transmission electron microscopy were performed to observe the histopathological changes.The expressions of key proteins in the NLRP3/Caspase-1 pathway of pyroptosis and the IL-1β,a downstream factor of pyroptosis in the liver were detected by immunohistochemistry,Western blot and ELISA.Results Compared with the control group,the cystic lesions on the surface of liver tissues in the infected group mice gradually increased and protruded from the liver surface with the extension of infection time.HE staining showed various pathological changes such as inflammatory cell infiltration and fibrous hyperplasia in the liver lesions to varying degrees.After 2 months of Em infection,transmission electron microscope observation showed that the cell membrane of hepatocytes were broken and discontinuous,conforming to the"punching"phenomenon of pyroptosis.The results of ELISA showed that the concentration of IL-1βin liver homogenate of mice after 1,2,3 and 5 months of Em infection were significantly higher than that of the control group,and the difference was statistically significant(F=127.2,P<0.05).Immunohistochemical examination showed that the positive cell ratios of Caspase-1 and NLRP3 in liver of mice infected with Em at 1,2,3 and 5months,were higher than that of the control group,and the difference were statistical

关 键 词：多房棘球蚴病 肝脏 细胞焦亡 

分 类 号：R532.32[医药卫生—内科学] R-332[医药卫生—临床医学]