荧光原位明胶酶谱法对肝组织明胶酶活性的检测  

Detection for gelatinase activity of liver using flourescent in situ zymography

作　　者：陈倩[1] 沈丽[1] 何琦麟[1] 陶艳艳[1] 刘成海[1] 

机构地区：[1]上海中医药大学附属曙光医院肝病研究所,上海市201203

出　　处：《世界华人消化杂志》2008年第15期1607-1611,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30772869~~

摘　　要：目的:建立荧光原位明胶酶谱法,观察纤维化和急性损伤肝组织明胶酶活性表达特点.方法:复制二甲基亚硝胺大鼠肝纤维化模型与N-乙酰半乳糖胺/脂多糖急性肝损伤模型,取其肝组织冰冻切片,将绿色荧光明胶底物附着于肝组织冰冻切片上,置于酶反应缓冲液中避光孵育8-24h;再以Hoechst液复染细胞核,在荧光显微镜下分别以蓝光激发拍摄明胶酶绿色荧光和紫光激发拍摄细胞核蓝色荧光,并将图像重叠.结果:成功建立荧光原位明胶酶谱法.正常大鼠肝组织仅在肝窦周围少量明胶酶表达,纤维化及急性肝损伤肝组织肝窦处明胶酶活性增强,纤维化肝组织纤维间隔处明胶酶活性也较强.结论:荧光原位明胶酶谱法具有灵敏,直观,简便的优点,可较好分析肝组织明胶酶活性水平与表达位置,对研究肝脏病理与药理具有重要意义.AIM： To establish fluorescent in situ zymography for gelatinase activity of liver and observe the feature of gelatinase activity in liver tissues with fibrosis and acute injury. METHODS： Hepatic fibrosis of rats was induced by administration of dimethylnitrosamine （DMN） intraperitoneally and acute liver injury of mice by injection of GalN and lipopolysaccharide （LPS） intraperitoneally. Gelatin substrate of green fluorescence was mounted on cryostat sections of liver tissues from the above two models, incubated for 8-24 h, and then the nuclei were counterstained with Hoechst. The green fluorescence of gelatinase and blue fluorescence of nuclei were observed under fluorescence microscope, and the images were overlapped. RESULTS： Fluorescent in situ zymography for gelatinase activity was established successfully. With this method, a low level of gelatinase activity was detected in the sinus hepaticus of normal liver sections; however, it was enhanced in the sinus hepaticus of liver sections with fibrosis and acute injury. Gelatinase activity in the fibrous septum of liver fibrosis sections was strong. CONCLUSION： Flourescent in situ zymography is sensitive, directviewing and convenient in the detection of gelatinase activity level and position and of great significance for the studies on pathology and pharmacology of liver.

关 键 词：明胶酶活性 荧光原位明胶酶谱法 肝脏 

分 类 号：R575[医药卫生—消化系统]