金属硫蛋白表达在C57BL/6J小鼠肝细胞癌发生中的变化及意义

Changes and significance of metallothionein expression during hepatocarcinogenesis in C57BL /6J mice

目的了解金属硫蛋白（MTs）基因表达水平在小鼠肝细胞癌（HCC）发生过程中的动态变化及其规律,探讨MTs在HCC发生中的重要意义。方法将125只5~8周龄雄性C57BL/6J小鼠随机分为正常对照组和HCC模型组。模型组小鼠于第1、2周分别腹腔注射一次二乙基亚硝胺（100 mg/kg,50 mg/kg）,第3周起灌胃给予乙醇（53%,5 m L/kg,5 d/周）,直至35周;正常组始终给予等量灭菌自来水灌胃。分别于实验1、3、9、13、24及35周末处死小鼠,收集肝组织样本并计算肝脏指数。采用组织病理学HE、Masson及网状纤维染色检测肝组织损伤及HCC发生情况,采用酶联免疫吸附法检测肝组织匀浆丙二醛（MDA）的活性;采用实时荧光定量PCR分析MTs转录水平。结果模型组小鼠可见进行性的肝脏病变,35周末时,肝脏质地显著变硬,表面可见大小不等结节形成,约50%小鼠可见肝细胞异常核分裂像和异常的肝板结构等HCC发生病理改变,具有显著增加的肝脏指数;不同时段均表现出增加的MDA活性;实验13周前各时段具有显著增加的MTs mRNA水平,24周后可见III级纤维化形成,且MTs mRNA水平显著下降,甚至低于正常水平。结论小鼠肝组织MTs mRNA水平从损伤初期的显著增加直至显著纤维化后的低表达变化,表明MTs表达下调与HCC发生有关。

Objective To investigate the dynamic changes of metallothionein（ MTs） gene expression and explore the important significance of MTs during hepatocarcinogenesis. Methods One hundred and twenty-five SPF 5- 8-week old male C57 BL /6J mice were randomly divided into control group and model group. Diethylnitrosamine（ DEN） was given to the mice at a dose of 100 mg / kg,ip,and 50 mg / kg,ip,in the first and next week,respectively. The mice were given ethanol（ 53%,5 m L / kg / day,5 days / week） from the third week of experiment till 35 weeks. At 1,3,9,13,24 and 35 weeks of the experiment,liver samples were taken for histopathological examination of liver damages and incidence of HCC.The liver index and malondialdehyde（ MDA） of liver homogenate were determined. All liver tissue samples were examined by histopathology using hematoxylin and eosin（ HE）,Masson and reticular fiber staining. Real-time RT-PCR was used to analyze the mRNA expression level of liver metallothionein-1 /2（ MT-1 /2） in different periods. Results Progressive liverdamages in model group mice were identified in different periods. Hepatocytes abnormal tission and abnormal liver plate structure,architecture often characteristic of HCC could be seen in approximately 50% of mice at 35 weeks. In addition to these,a higher liver index also were seen at 35 weeks. Increased MDA levels in the mouse liver tissues were observed in each stage. Real-time RT-PCR analysis showed that significantly increased transcription of MT-1 and MT-2 at 1,3 and 9weeks,then gradually declined and even below the normal level. Conclusions MTs gene expression levels in mouse liver tissues are changed from significantly increased in the early stage of injury to decreased expression combined with distinct fibrosis. Our findings further demonstrate that the down-regulation of MTs level is closely correlated with hepatocarcinogenesis.