生长抑素受体-2基因逆转上皮-间充质转化抑制肝癌细胞侵袭转移机制的研究  

The mechanism of somatostatin receptor subtype 2 reversed hepatocellular carcinoma cells epithelial mesenchymal transition and inhibited invasion and metastasis

作　　者：郑建伟[1] 张晓梅 张春霞 李雁[4] 易继林[1] 秦仁义[1] 吴在德[1] 薛新波[1] 申铭[5] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外科,武汉430030 [2]武汉市第五医院消化内科 [3]信阳市中心医院妇产科 [4]武汉大学附属中南医院肿瘤科 [5]华中科技大学同济医学院附属同济医院胆胰外科,武汉430030

出　　处：《中华实验外科杂志》2013年第10期2036-2039,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30872510）;湖北省自然科学基金资助项目（2008CDB127）

摘　　要：目的 观察生长抑素受体-2（SSTR2）基因对肝癌细胞株MHCC-97H侵袭转移及上皮-间充质转化（EMT）特性的影响.方法 构建慢病毒3FLAG-puromycin-LV及SSTR2-LV,转染肝癌细胞株MHCC-97H,噻唑蓝（MTT）、免疫荧光检测转染效率.采用Transwell侵袭实验和迁移实验分别检测未转染细胞（空白对照组）、转染3FLAG-puromycin-LV细胞（阴性对照组）和转染SSTR2-LV细胞（实验组）侵袭转移能力的强弱.镜下观察转染前后细胞形态的变化,免疫荧光检测转染前后细胞上皮表型蛋白和间质表型蛋白的定位和表达,实时定量聚合酶链反应（Real-time PCR）检测转染后细胞SSTR2的表达,Westem blot检测E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）蛋白的表达.结果 SSTR2-LV可以有效转染肝癌细胞株MHCC-97H,稳定表达SSTtR2的mRNA和蛋白.转染SSTR2-LV的肝癌细胞株MHCC-97H较空白对照组和阴性对照组侵袭迁移能力明显受到抑制（P＜0.05）.镜下观察肝癌细胞株MHCC-97H转染SSTR2-LV后,由成纤维细胞样、排列分散,转变为细胞排列紧密如铺路石.免疫荧光染色结果可见肝癌细胞株MHCC-97H转染后细胞膜表达的E-cadherin荧光由胞质转移至细胞周边浓聚,而Vimentin的荧光强度较前下降.采用Western blot进行蛋白定量分析,转染后MHCC-97H细胞上皮表型标志性蛋白E-cadherin、β-连环蛋白（β-catenin）表达量增高,间质表型标志性蛋白N-cadherin、Vimentin表达量减少,差异具有统计学意义（P＜0.05）.结论 转染再表达SSTR2MHCC-97H细胞可以增加上皮表型蛋白表达,减少间质表型蛋白,从而逆转肝癌细胞EMT,导致肿瘤细胞侵袭能力减弱.Objective To investigate the effect of the somatostatin receptor subtype 2 （SSTR2） on the migration and invasion of human Hepatocellular carcinoma cells MHCC-97H and the change of epithelial-mesenchymal transition （EMT）.Methods Constructed 3FLAG-puromycin-LV and SSTR2-LV and transfected hunman Hepatocellular carcinoma cells （MHCC-97H） respectively.The expression of SSTR2 was detected by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot.The migration and invasion of Non-transfected （blank control group）,3FLAG-puromycin-LV transfected （negative control group） and SSTR2-LV （experimental group） transfected MHCC-97H cells was detected by Transwell invasion assay and Transwell migration assay.The changes in morphology of cells were observed by optical microscopy.The localization and expression of the epithelial markers and the mesenchymal markers were assayed by immunofluorescence and Western blotting.Results The stable mRNA and protein expression of SSTR2 was detected in the cells transfected with SSTR2-LV.The migration and invasion was restrained in the experimental group compared with control groups （P ＜ 0.05）.Transfection of SSTR2-LV induced a morphologic change of MHCC-97H cells from fibroblastic shape with cell scattering to a paving stone structure with cell-cell adhesion.In SSTR2-LV-transfected cells,the localization of E-cadherin was altered from the cytoplasm to cell-cell contacts and the level of Vimentin decreased.Moreover,the expression of the epithelial marker,E-cadherin and β-catenin,were increased; the expression of the mesenchymal marker,N-cadherin and Vimentin,were reduced.Conclusion MHCC-97H re-expression of SSTR2 induced the increasing expression of epithelial marker and the decrease expression of mesenchymal marker reversed HCC EMT then suppressed the HCC cells migration and invasion.

关 键 词：生长抑素受体-2 上皮-间充质转化 肝细胞癌 侵袭 转移 

分 类 号：R735.7[医药卫生—肿瘤]