中文摘要：

目的探讨胃泌素对结肠癌细胞CoLo320WT中粘着斑激酶（FAK）通路下游E-钙粘蛋白／β-连环蛋白（E-cadherin／β-catenin）复合物分布的影响；方法脂质体转染表达胃泌索受体CCK-2R的pCR3．1／OR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，转染成功命为CoLo320WT。应用10^-8mmol／L 胃泌素（G17）以时间梯度（0h、1h、6h、12h、24h、48h）干预CoLo320WT细胞，同时应用10^-6mmol／L胃泌素受体拮抗剂L365，260干预CoLo320WT细胞30min，再予10^-8mmol／L胃泌素干预。采用免疫印迹法检测磷酸化的FAK Tyr397和总FAK的表达。采用免疫共沉淀和免疫印迹法检测CoLo320WT中TX-100溶解和未溶部分中的E-钙粘蛋白和β-连环蛋白的表达。用免疫细胞化学法观察E-钙粘蛋白和β-连环蛋白的在胞膜、胞质和胞核的分布。结果随着胃泌素干预时间的延长，细胞中磷酸化的FAK Tyr397的表达量呈增加趋势，12h达最大值。胃泌素受体拮抗剂L365，260阻断后磷酸化的FAK Tyr397表达减少。而胃泌素对总FAK没有明显影响。TX-100可溶性部分中E-钙粘蛋白和酽连环蛋白的量在胃泌素干预后表达减少，拮抗剂L365，260阻断后又增加。而TX-100不溶解部分中表达却相反。免疫细胞化学观察到在胃泌素干预下CoLo320WT细胞中E-钙粘蛋白和β-连环蛋白的分布发生胞质和胞核转移。结论胃泌素与其受体CCK-2受体结合，磷酸化的FAK Tyr397、激活FAK通路进而影响结肠癌细胞中E-钙粘蛋白和β-连环蛋白的分布，促进结肠癌细胞侵袭和转移。

英文摘要：

Objective To explore the effect of gastrin17 on E-cadherin/β-catenin complex, a down stream effector of FAK pathway, in colonic carcinoma cell line CoLo320WT. Methods pCR3. 1/GR plasmid expressing gastrin receptor CCK-2R was transfected into colonic carcinoma cell line CoLo320 by Lipofectamine^TM 2000 and expressing stably CCK-2R clones was screened by G418. The expression levels of gastrin receptor of CoLo320 and the transfected ceil line CoLo320WT were assayed by RT-PCR. CoLo320WT cells were treated by 10^- 8mmol/L gastrin17 at distinct time points （0 hr, 1 hr, 6 hr, 12 hr, 24 hr, 48 hr）,whilst treated by 10 ^-6 mmol/L L365, 260 （gastrin17 receptor blocker） simultaneously for 30 minutes and then treated by gastrin17 again for 12 hr. Expression levels of phosphorylated FAK Tyr397 and total FAK in CoLo320WT under gastrin17 intervention were detected by Western blot. Expression levels of E-cadherin and β-catenin complex in TX-100 solution fraction and TX-100 insolution fraction of CoLo320WT cells were detected by coimmunoprecipitation and Western blot. Distribution of E-cadherin and β-catenin in CoLoWT320 were observed by immunocytochemistry. Results Phosphorylated FAK Tyr397 expression in CoLo320WT cells increased in time-dependent fashion under gastrin17 intervention and peaked at 12 hour after intervention, while decreased by L365, 260 inhibition. But gastrin17 had no effect on total FAK in CoLo320WT cells. Expresion levels of E-cadherin and β-catenin cornplex in TX- 100 solution fraction were decreased apparently, but increased again after 1.365, 260 block ing. On the contrary, the expression levels of E-cadherin and β- catenin complex in TX-100 solution frac tion differed from that in TX -100 solution. Cytochemistry observation had revealed that E cadherin and catenin transferred from cell membranes into endochylemas, nuclei and cytoskeleton under gastrin17 in tervention. Conclusions Gastrin17 affected significantly the distribution of E-cadherin/β-catenin complex in CoLo320WT by phosphorating