hFRNK基因转移对结肠癌细胞中E-钙黏素／β-连环素复合物的影响  

Effects of hFRNK on E-cadherin/β-catenin in colon cancer cells in vitro

作　　者：曹俊[1] 于皆平[1] 刘超红[2] 陈新文[2] 刘嵩 罗和生[1] 于红刚[1] 

机构地区：[1]武汉大学人民医院消化内科,430060 [2]中国科学院武汉病毒所病毒学国家重点实验室 [3]武汉市第一人民医院消化内科

出　　处：《中华肿瘤杂志》2007年第5期346-350,共5页Chinese Journal of Oncology

基　　金：国家自然科学基金资助项目（30470782）

摘　　要：目的研究hFRNK基因对结肠癌细胞Colo320WT中E-钙黏素（E-cadherin）／β-连环素（β-catenin）复合物的影响。方法利用AdEasy^TM系统在大肠肝菌内同源重组构建表达人FRNK基因的腺病毒载体pAdhFRNK。脂质体转染pCR3．1／GR质粒于结肠癌细胞Colo320中,G418筛选出稳定表达CCK-2R的阳性克隆,RT-PCR鉴定。用10^-8 mol／L胃泌素干预Colo320WT细胞12 h和pAdhFRNK体外感染Colo320WT细胞2 d后,再用10^-8 mol／L的胃泌素干预细胞12 h,然后用免疫共沉淀观察TX-100可溶性部分和不溶性部分的E-cadherin和β-catenin的表达。利用细胞化学方法观察E-cadherin和β-catenin在Colo320WT细胞中的分布情况。结果在胃泌素干预12 h后的TX-100可溶性部分中,E-cadherin和β-catenin的表达量明显降低,而TX-100不溶解部分表达增加。pAdhFRNK感染胃泌素干预后的细胞,在TX-100可溶性部分中,E-cadherin和β-catenin的表达量增加,而TX-100不溶性部分中表达降低。细胞化学方法显示,E-cadherin和β-catenin在胃泌素干预12 h后,由胞膜向胞内和胞核转移,而胃泌素干预pAdhFRNK感染后的细胞中,两者分布又逆转。结论hFRNK基因可明显对抗外源性胃泌素引起Colo320WT细胞中E-cadherin和β-catenin的分布异常,其机制可能是通过阻断FAK磷酸化及其通路而实现。Objective To explore the effects of human FRNK gene on E-cadherin/β-catenin complex in colon cancer cell line Colo320WT cells stimulated with extrinsic gastrin17. Methods AdEasy^TM system was used to construct pAdhFRNK expressing human FRNK gene by recombination in E. coli. BJ5283. pCR3.1/GR plasmid expressing gastrin receptor CCK-2 was transfected into colon cancer cell line Colo320 cells by Lipofectamine^TM 2000 and expressing stably CCK-2R clones were screened by G418 （500μg/ml）. The expression levels of gastrin receptor in Colo320 cells and the transfected Colo320WT cells were assayed by RT-PCR. Colo320WT cells were treated by 10^-8 mol/L gastrin17 for 12 h; and after Colo320WT cells were infected by pAdhFRNK （MOI： 100） for 2 d the cells were treated by gastrinl7 for 12 h again. The expression levels of E-cadherin and β-catenin in TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells were assayed by co-immunoprecipation and Western blot. E-cadherin and β-catenin＇s distribution in Colo320WT cells were detected by immunocytochemistry. Results When 10^-8 mol/L gastrin17 stimulated Colo320WT cells for 12 h, the expression levels of E-cadherin and β-eatenin in TX-100- soluble fraction decreased apparently, while the expression levels of E-cadherin and β-catenin in TX-100- insoluble fraction increased markedly. When pAdhFRNK infected Colo320WT cells for 2 d and 10^-8 mol/L gastrin17 treated the cells for 12 h, the expression levels of E-cadherin and β-catenin in TX-100-soluble fraction increased apparently again, and the expression levels of E-cadherin and β-catenin in TX-100- insolutble fraction decreased markedly. Immunocytochemtry showed that the distribution of E-cadherin and β-catenin was translocated from plasma membrane into cytoplasm and nucleus in the cells stimulated with gastrin17, and after the cells were infected with pAdhFRNK and stimulated by gastrin17 again. β-catenin was mainly observed in cytoplasm and little nuclear immunoreactivity. Conclusion An adenovir

关 键 词：腺病毒载体 基因治疗 结肠肿瘤 E-cadherin/β-catenin复合物 

分 类 号：R686[医药卫生—骨科学]