肝脏特异性敲除核因子-κB必需调节蛋白基因对Myc过表达诱导的小鼠原发性肝癌发生发展的影响  

Effect of liver knockout of nuclear factor-κB essential modulator on the development of mouse hepatocarcinogenesis induced by Myc overexpression

作　　者：贺佳佳 丁昀 韦炜[1] 张楚[1] 吴骏[1] 胡文蔚[1] 季枚[1] 吴昌平[1] 蒋敬庭[1] He Jiajia;Ding Yun;Wei Wei;Zhang Chu;Wu Jun;Hu Wenwei;Ji Mei;Wu Changping;Jiang Jingting(Cancer Biology Diagnosis and Treatment Center of the Third Affiliated Hospital of Soochow University,Jiangsu Engineering Research Center for Tumor Immunotherapy,Institute of Cell Therapy,Soochow University,Changzhou 213003,China)

机构地区：[1]苏州大学附属第三医院肿瘤生物诊疗中心,江苏省肿瘤免疫治疗工程技术研究中心,苏州大学细胞治疗研究院,常州213003

出　　处：《中华实验外科杂志》2020年第10期1804-1807,共4页Chinese Journal of Experimental Surgery

基　　金：国家重点研发资助项目(2018YFC1313400);国家科技支撑计划资助项目(2015BAI12B12);国家自然科学基金海外及港澳学者合作研究基金项目(31729001);国家自然科学基金(81972869);江苏省重点研发计划专项资金项目(BE2018645);常州市科技计划项目(CJ20190088、CJ20190089)。

摘　　要：目的:探讨IκB激酶(IKK)/核因子-κB(NF-κB)信号通路对Myc过表达诱导的小鼠原发性肝癌发生发展的影响。方法:利用转基因小鼠将LAP-tTA小鼠与Alb-cre小鼠杂交,再与NEMO f1(NEMO为NF-κB必需调节蛋白)小鼠杂交,以产生LAP-tTA+/Alb-cre+/NEMO fl/wt小鼠,最后与tetO-Myc+小鼠杂交。本实验包括4组:(1)WT小鼠;(2)NEMOΔLPC小鼠(肝细胞中NEMO敲除,无Myc过表达);(3)Myc LAP-tTA小鼠(Myc过表达,无NEMO敲除);(4)Myc LAP-tTA/NEMOΔLPC小鼠(肝脏Myc过表达和NEMO敲除)。记录小鼠的生存曲线,观察肝脏大体形态。苏木精-伊红(HE)染色观察肝组织病理结构,免疫组织化学染色、蛋白质印迹法(Western blot)检测肝脏肿瘤指标及肝祖细胞指标、定量聚合酶链反应(qPCR)方法检测相关蛋白的mRNA水平。使用t检验比较独立样本组与相应组。结果:小鼠带瘤生存曲线显示Myc LAP-tTA/NEMOΔLPC小鼠中位生存期明显短于Myc LAP-tTA小鼠(中位生存期M/P 5056 d比83 d,P<0.01);Myc LAP-tTA/NEMOΔLPC小鼠对比Myc LAP-tTA小鼠,谷草转氨酶[(739.40±35.61)U/L比(441.50±78.79)U/L,t=2.464,P<0.05]、碱性磷酸酶[(3142.0±287.6)U/L比(1702.0±278.8)U/L,t=3.129,P<0.01]、γ-谷氨酰基转移酶[(101.70±12.82)U/L比(37.31±9.34)U/L,t=3.927,P<0.01]和总胆红素[(1.281±0.232)mg/dl比(0.618±0.051)mg/dl,t=3.889,P<0.01]均显著升高,且差异有统计学意义;Western blot检测显示细胞角蛋白19(CK19)、性别决定区Y框蛋白9(SOX9)水平在Myc LAP-tTA/NEMOΔLPC较Myc LAP-tTA组小鼠肝脏亦明显升高;肝祖细胞标志物如角蛋白(CK19,4.336±1.970比0.680±0.234,t=1.843,P<0.01)、CK7(3.884±0.338比2.370±0.525,t=2.422,P<0.01)、甲胎蛋白(AFP,3614.0±2070.0比1399.0±319.9,t=1.057,P<0.01)、上皮细胞黏附分子(EpCAM)的mRNA水平在Myc LAP-tTA/NEMOΔLPC较Myc LAP-tTA组小鼠肝脏显著增高(7.900±0.997比3.084±0.711,t=3.943,P<0.01)。结论:肝细胞中特异性敲除NEMO从而抑制经典的IKK/NF-κB信号通路,可促进肝肿瘤的发生发展Objective To investigate the role of IκB kinase(IKK)/nuclear factor-κB(NF-κB)signaling pathway in the development of Myc-induced mouse hepatocellular carcinoma and potential mechanisms.Methods Transgenic mice were used in the study.LAP-tTA mice were crossed with Alb-cre transgenic mice,and the double transgenic offsprings were crossed to nuclear factor-κB essential modulator(NEMOf1)mice to generate LAP-tTA+/Alb-cre+/NEMOfl/wt mice,which lastly were crossed to tetO-Myc+mice.Four groups of animals were included:(1)WT mice(no expression of Myc and no NEMO ablation),(2)NEMOΔLPC mice(specific deletion of NEMO in liver parenchymal cells but no expression of Myc),(3)MycLAP-tTA mice(only overexpression of Myc,no ablation of NEMO),and(4)MycLAP-tTA/NEMOΔLPC mice(both overexpression of Myc and ablation of NEMO).Kaplan-Meier survival curve was recorded,and liver was observed both macroscopically and microscopically.Hematoxylin and eosin(HE)staining was used to observe the pathology of liver tissues.Immunohistochemical staining,Western blotting and quantitative polymerase chain reaction(qPCR)were performed to detect liver tumor markers and liver progenitor cell markers.Results The Kaplan-Meier survival curve of mice showed that the median survival time of MycLAP-tTA/NEMOΔLPC mice was significantly shorter than that of MycLAP-tTA mice,and the difference was statistically significant(Median survival time M/P50,56 d vs.83 d,P<0.05).The serum levels of aspartate aminotransferase[(739.40±35.61)U/L vs.(441.50±78.79)U/L,t=2.464,P<0.05],alkaline phosphatase[(3142.0±287.6)U/L vs.(1702.0±278.8)U/L,t=3.129,P<0.01],γ-glutamin transferase[(101.70±12.82)U/L vs.(37.31±9.34)U/L,t=3.927,P<0.01]and total bilirubin[(1.281±0.232)mg/dl vs.(0.618±0.051)mg/dl,t=3.889,P<0.01]were significantly increased in livers of MycLAP-tTA/NEMOΔLPC mice compared to MycLAP-tTA mice(P<0.05);Western blotting analysis showed that cytokeratin 19(CK19)and sex determining region Y box 9(SOX9)protein levels in MycLAP-tTA/NEMOΔLPC mice were significantl

关 键 词：核因子-κB必需调节蛋白 MYC 原发性肝癌 肝祖细胞 

分 类 号：R735.7[医药卫生—肿瘤]