NDUFA13失活在诱导小鼠自发性肝炎病理进程中的作用及机制  

Pathogenic role of NDUFA13 inactivation in spontaneous hepatitis in mice and the mechanism

作　　者：徐小惠 李锐 曾欣 王欣 薛炳乾 黄道超[1] 黄轶[1] XU Xiaohui;LI Rui;ZENG Xin;WANG Xin;XUE Bingqian;HUANG Daochao;HUANG Yi(Institute of Pediatrics,Children's Hospital of Chongqing Medical University,Chongqing Key Laboratory of Child Infection and Immunity//Key Laboratory of Child Development and Disorders of Ministry of Education//National Clinical Research Center for Child Health and Disorders//China International Science and Technology Cooperation Base of Child Development and Critical Disorders,Chongqing 400014,China)

机构地区：[1]重庆医科大学附属儿童医院儿科研究所//重庆市儿童感染与免疫重点实验室//儿童发育疾病研究教育部重点实验室//国家儿童健康与疾病临床医学研究中心//儿童发育重大疾病国家国际科技合作基地,重庆400014

出　　处：《南方医科大学学报》2021年第1期55-63,共9页Journal of Southern Medical University

基　　金：国家自然科学基金(30701004);重庆市渝中区基础研究与前沿探索项目(20190106)。

摘　　要：目的构建线粒体蛋白(NDUFA13)基因肝特异性敲除小鼠,探讨NDUFA13表达失活在小鼠自发性肝炎病理进程中的作用及可能机制。方法采用Cre/loxP转基因技术,将NDUFA13 flox基因编辑小鼠NDUFA13fl/fl与Alb-Cre转基因小鼠杂交,得到肝脏特异性NDUFA13杂合敲除(NDUFA13fl/-;Alb-Cre)小鼠。经PCR扩增及琼脂糖凝胶电泳,对鼠尾与肝脏DNA基因进行分型鉴定。以同窝NDUFA13fl/fl小鼠为对照,将肝脏特异性NDUFA13fl/-小鼠作为研究对象,10只/组,分别在4周龄、2年龄时各处死5只,获取小鼠肝组织样本,通过HE染色分析肝组织病理变化,免疫组化分析NDUFA13、NF-κB/p65、NF-κB/p-p65及炎症小体NLRP3的表达,ROS染色试剂盒分析细胞总ROS及线粒体ROS水平;免疫组化与免疫荧光分析免疫细胞标志物CD45、MPO、F4/80以及炎症因子IL1β、IL33的表达情况。结果PCR鉴定结果显示成功建立肝特异性NDUFA13杂合敲除小鼠;HE染色显示4周龄与2年龄NDUFA13fl/-小鼠肝组织均损伤严重;免疫组化检测显示,4周龄与2年龄NDUFA13fl/-小鼠NDUFA13蛋白表达显著下调(P<0.05),NF-κB信号分子p65、p-p65(Ser536)及炎症小体NLRP3蛋白表达显著增加(P<0.05);ROS测定发现NDUFA13基因杂合敲除导致小鼠肝脏细胞总ROS、线粒体ROS水平均显著增加;免疫组化与免疫荧光结果显示,肝组织CD45、MPO、F4/80等炎症细胞标志物的聚集以及IL1β、IL33等炎症因子的分泌均明显增多(P<0.05)。结论NDUFA13蛋白表达失活可诱导小鼠自发性肝炎病理表型,其机制可能与活化的ROS/NF-κB/NLRP3信号通路相关。Objective To investigate the role of NDUFA13 inactivation in the pathogenesis of spontaneous hepatitis in mice and explore the possible mechanisms.Methods Hepatocyte-specific NDUFA13 knockout(NDUFA13fl/-)mice were generated by intercrossing NDUFA13fl/fl and Alb-Cre mice based on Cre/loxP transgenic technology,and tail and liver DNA of the mice was genotyped by PCR analysis.Ten NDUFA13fl/-mice and 10 littermate control NDUFA13fl/fl mice were housed,and in each group,5 mice were euthanized at the age of 4 weeks and the other 5 at two years for pathological examination of the liver tissues with HE staining.Immunohistochemistry was used to verify the expression levels of NDUFA13,NF-κB/p65,NF-κB/p-p65 and inflammasome NLRP3.The total intracellular ROS and mitochondrial ROS levels were measured with a ROS staining kit.The expressions of the inflammatory cell markers(CD45,MPO,and F4/80)and inflammatory cytokines(IL1βand IL33)in the liver were detected with immunohistochemistry and immunofluorescence assay.Results Liver-specific NDUFA13 heterozygous knockout mice were successfully constructed as verified by PCR results.HE staining revealed severe liver damage in both 4-week-old and 2-year-old NDUFA13fl/-mice as compared with their littermate controls.Immunohistochemistry showed a significant decrease of NDUFA13 expression in both 4-week-old and 2-year-old NDUFA13fl/-mice(P<0.05).The expression levels of NF-κB signals p65,p-p65 and NLRP3 inflammasomes were all significantly increased in NDUFA13fl/-mice(P<0.05).The total intracellular ROS and mitochondrial ROS levels in NDUFA13fl/-mice were also significantly increased.NDUFA13 knockout obviously promoted the expression of the inflammatory cell markers(CD45,MPO and F4/80)and the secretion of IL-1βand IL-33 in the liver tissue of the mice(P<0.05).Conclusion Hepatocytes-specific NDUFA13 ablation can trigger spontaneous hepatitis in mice possibly mediated by the activation of ROS/NF-κB/NLRP3 signaling.

关 键 词：NDUFA13 基因敲除小鼠模型 NLRP3 自发性肝炎 

分 类 号：R575.1[医药卫生—消化系统]