摘要目的 研究高迁移率族蛋白1 (HMGB1)对二乙基亚硝胺诱发C57BL/6小鼠肝癌形成的促进作用及其机制.方法 HMGB1loxp/loxp/Alb-Cre+/-为肝脏特异性敲除HMGB1基因(KO)的小鼠,同窝出生的HMGB1loxp/1oxp/Alb-Cre-/-,HMGB1loxp/WT/Alb-Cre+/-和HMGB1loxp/WT/Alb-Cre-/-为野生型(WT)小鼠.分别取6只出生12d的雄性WT和KO的小鼠,一次性腹腔注射二乙基亚硝胺25 mg/kg.6个月后取肝组织HE染色评价病理学改变并统计各组小鼠肝癌发生率;取各组小鼠血清样本测定丙氨酸转氨酶水平;免疫组织化学染色检测两组小鼠瘤组织内HMGB1蛋白的表达和胞内定位情况;蛋白质印迹法(Western blot)检测两组小鼠瘤组织内线粒体生物合成相关基因的表达隋况;RT-PCR检测两组小鼠瘤组织和正常肝组织线粒体DNA拷贝数.组内数据比较采用t检验,组间比较采用单因素方差分析.结果 与WT小鼠相比,KO小鼠肝/体质量比明显下降(t=2.634,P=0.022 5);两组小鼠血清丙氨酸转氨酶水平均有升高,但差异无统计学意义(t=0.4062,P=0.693 2).WT小鼠肝脏表面可见较多大小不一的灰白色结节,组织学类型为肝细胞癌,不同基因型WT小鼠肝癌发生率差异无统计学意义(P＞ 0.05);KO小鼠的肝癌发生率明显降低(t=8.521,P＜0.001).和WT小鼠瘤组织相比,KO小鼠瘤组织内HMGB1和线粒体生物合成相关基因过氧化物酶体增殖物激活受体-γ共激活因子-1α和核呼吸因子1表达量显著下降(t值分别为6.238、4.852,P值分别为0.033 5、0.041),线粒体DNA拷贝数明显降低(t=9.211,P＜0.01);WT小鼠瘤组织线粒体DNA拷贝数明显高于正常肝组织(t=8.305,P=0.014 2).结论 HMGB1通过诱导线粒体生物合成促进二乙基亚硝胺诱发肝癌的形成.

abstractsObjective To study the role of high-mobility group protein 1 (HMGB1) in the promotion of diethylnitrosamine-induced liver cancer formation in C57BL/6 mice and its mechanism.Methods HMGB1loxp/loxp/Alb-Cre+/-were used as a liver-specific knockout (KO) of HMGB1 gene in mice.HMGB1loxp/loxp/Alb-Cre-/-,HMGB1loxp/WT/Alb-Cre+/-and HMGB1loxp/WT/Alb-Cre-/-born in the same litter were wild-type mice.Six 12-day-old male WT and KO mice were separated and given a single intraperitoneal injection of diethylnitrosamine (25 mg/kg).Six months later,HE staining was used to evaluate the histopathological changes and then the incidence of liver cancer in each mice group was calculated.Serum samples were taken from each mice group to determine alanine aminotransferase levels.Immunohistochemical staining was used to detect the expression and intracellular localizations of HMGB1 protein status in tumor tissue of the two groups of mice.Western blot was used to detect the expressional condition of mitochondrial biogenesis in tumor tissue of the two groups of mice.RT-PCR was used to detect mitochondrial DNA copy number of tumor tissue and normal liver tissue in the two groups of mice.Intra and inter group data comparison was compared using t-tests and one one-way analysis of variance.Results Compared with WT mice,the liver/body weight ratio of KO mice was decreased significantly (t =2.634,P =0.0225).Serum alanine aminotransferase levels in both groups of mice were increased,and the difference was not statistically significant (t =0.4062,P =0.6932).There were many visible gray-white nodules of different sizes on the liver surface of WT mice,and the histological type was hepatocellular carcinoma.There was no statistically significant difference in the incidence of liver cancer among different genotypes of WT mice (P ＞ 0.05).The incidence rate of liver cancer in KO mice was significantly reduced (t =8.521,P ＜ 0.001).Compared with WT mice,the expression levels of HMGB1 and mitochondrial biogenesis (PGC-1α and NRF1) was significantly reduced (t =6.238,4.852,P =0.0335,0.041) in tumor tissue of KO mice.Mitochondrial DNA copy number was decreased significantly (t =9.211,P ＜ 0.01).Mitochondrial DNA copy number in tumor tissue of WT mice was significantly higher than that in normal liver tissue (t =8.305,P =0.0142).Conclusion HMGB1 promotes the formation of diethylnitrosamine-induced liver cancer by inducing mitochondrial biogenesis.