人结肠癌细胞胃泌素-黏着斑激酶通路的研究

目的研究胃泌素对人结肠癌细胞信号分子黏着斑激酶(FAK)酪氨酸磷酸化和蛋白质表达的影响。方法使用胆囊收缩素2受体(CCK2R)的真核表达载体pCR3.1/CCK2R,转染人结肠癌细胞株Colo320,上调胃泌素作用;使用胃泌素拮抗剂下调胃泌素作用。使用胃泌素按浓度和时间梯度刺激细胞,以免疫沉淀和蛋白质印迹法检测FAK酪氨酸磷酸化和蛋白质表达情况。结果胃泌素能够引起FAK酪氨酸磷酸化,具有时间和剂量依赖性;CCK2R表达上调可以增强此作用;胃泌素拮抗剂具有相反作用。结论FAK是胃泌素发挥效应的下游信号分子,以酪氨酸磷酸化的形式发挥作用。胃泌素CCKRFAK信号通路在胃泌素引起的肿瘤细胞增殖过程中发挥重要作用。

A study of gastrin-focal adhesion kinase signal pathway in human colon cancer cells

Objective The study investigated the effect of gastrin on tyrosine phosphorylation and protein expression of focal adhesion kinase (FAK) in human colon cancer cells.Methods The eukaryotic plasmid that expresses cholecystokinin 2 receptor(CCK_2R) stably, named pCR3.1/CCK_2R,was transfected into Colo320 to construct an up-regulated gastrin-CCK_2R signal pathway; the gastrin antagonist was used to down-regulate the signal pathway. Thus, including the normal control, there were three signal pathways with different CCK_2R levels. Different doses of gastrin were used to stimulate FAK in the different time. FAK tyrosine phosphorylation and FAK expression in different groups were detected by immunoprecipitation and Western-blotting assays, and analyzed with Labimage software. Results RT-PCR result showed that Colo320 transfected with CCK_2R had a mRNA level four times higher than that normal Colo320 did, and which suggested that up-regulated signal transduction pathway was constructed. After stimulated by gastrin with 0, 0.1, 1, 10 and 100 nmol/L, tyrosine phosphorylation levels of Colo320 were 24.0%,39.7%, 46.2%, 50.4% and 44.5%, and those of Colo320 transfected with CCK_2R were 24.6%, 70.7%, 90.1%, 100% and 88.6%. When incubated with gastrin at 0, 2.5, 5, 10 and 20 min, the tyrosine phosphorylation levels of Colo320 were 23.9%, 63.6%, 58.6%, 45.5% and 40.9%, and those of Colo320 transfected with CCK_2R were 24.5%, 84.6%, 100%, 98.6% and 97.9%. The increases of tyrosine phosphorylation in both Colo320 and Colo320 transfected with CCK_2R were dose dependence of gastrin. In Colo320, the time of phosphorylation had a tendency of exhaustion at 2.5 min; but in Colo320 transfected with CCK_2R, it was at 10 min. Gastrin had no effects on FAK protein expression in different cell groups. An up-regulated level of CCK_2R could enhance the effect of gastrin on FAK tyrosine phosphorylation. The gastrin antagonist showed an effect of competitive inhibition on tyrosine phosphorylation of FAK. Conclusions FAK is a signal transducer in downstream of CCK_2R; FAK exerts its functions by tyrosine phosphorylation, but dose not increase FAK protein.Gastrin-CCK_2R-FAK signal pathway is a pivotal one in the cell growth and proliferation caused by gastrin.