体外稳定表达DMBT1对胆囊癌细胞系GBC-SD趋化迁移的影响  

Effects of DMBT1 transfection into GBC-SD cell line on migration

作　　者：贺涛[1] 刘厚宝[1] 艾志龙[1] 锁涛[1] 童赛雄[1] 王炳生[1] 杨勇[2] 查锡良[2] 

机构地区：[1]复旦大学附属中山医院普外科,上海200032 [2]复旦大学附属中山医院卫生部糖蛋白重点实验室,上海200032

出　　处：《中华普通外科杂志》2010年第2期122-125,共4页Chinese Journal of General Surgery

基　　金：复旦大学青年科学基金（No：167）

摘　　要：目的建立稳定表达候选抑癌基因DMBT1（deleted in malignant brain tumours1）的胆囊癌细胞系GBC—SD，研究其迁移能力并探讨分子机制。方法以脂质体为介导在体外建立稳定表达DMBT1的胆囊癌细胞系。细胞分组：（1）GBC-SD／DMBT1为转染DMBT1组；（2）GBC．SD／Vector为转染空载体组；（3）GBC—SD为未处理组。免疫组化、Westernblot及RT-PCR检测DMBT1蛋白、mRNA表达，确证转染成功，划痕法与Transwell法检测细胞迁移能力，Westemblot检测E-钙粘蛋白、CD44V6、CD15等黏附转移分子表达。统计学采用配对或独立样本t检验，P〈0．01为差异有统计学意义。结果稳定转染后，实验组DMBT1蛋白及mRNA表达较对照组分别提高3．2倍、2．7倍；划痕法显示实验组细胞在迁移距离、迁移速率、迁移面积均显著小于对照组（P〈0．01）；Transwell法表明转染DMBT1后，迁移细胞数亦显著少于对照组（P〈0．01）；免疫印迹表明转染实验组较对照组E-钙粘蛋白表达上调，CD15表达量减少（P〈0．01），而CD44V6、α与β连环素、整合素击在转染前后表达量无明显差异。结论在体外成功建立稳定表达DMBT1的胆囊癌细胞系；DMBT1在体外过表达显著抑制GBC—SD的迁移，至少部分依赖于上调E-钙粘蛋白表达，下调CD15表达。Objective To establish the gallbladder carcinoma cell line GBC-SD with stable expression of DMBT1 （deleted in malignant brain tumours 1 ） and explore effects on its chemotaxis and migration. Methods The full-length expression clone of DMBT1 was transfected with Lipofectamine 2000 into GBC-SD which was subsequently screened with G418 filtration. Semi-quantitative reverse transcription- PCR, Western blot and immunocytochemistry were employed to determine the level of DMBT1 mRNA and protein. Scratch wound model and transwell assay were applied to evaluate alterations of cell migration ability. Western blot was used to detect variances in the levels of proteins relevant to invasion and migration, namely CD15, CD44V6, E-cadherin, αand α catenin, integrin α5 and β1. Results RT-PCR, Western blotting and immunocytochemistry found significant increase of DMBT1 mRNA and protein, GBC-SD/ DMBT1 vs GBC-SD/vector, P 〈 0. 01. Scratch wound model witnessed markedly lessened parameters of migration distance, area, and rate in GBC-SD/DMBT1, P 〈 0. 01, when compared with that of GBC-SD/ vector. Transwell assay confirmed great discrepancy in migration, between GBC-SD/DMBT1 and GBC-SD/ vector. Western blot detected decreased level of CD15 and increased level of E-cadherin whereas no significant difference was found in α andα catenin, integrin α5 and α1, and CDd4V6 expression, P 〈 0. 01, between GBC-SD/DMBT1 and GBC-SD/vector. Conclusion The cell model with stable expression of DMBT1 was successfully established in vitro; DMBT1 over-expression can significantly inhibit the migration of the gallbladder carcinoma cell line GBC-SD possibly because of an up-regulation of E-cadherin and down-regulation of CD15.

关 键 词：胆囊肿瘤 基因 转染 细胞迁移分析 

分 类 号：R735.8[医药卫生—肿瘤] R394[医药卫生—临床医学]