摘要
目的探讨胃泌素促进人大肠癌细胞侵袭力增加的分子机制。方法脂质体转染表达胃泌素受体(CCK-2R)的 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,逆转录 PCR 和免疫印迹鉴定,转染成功命名为 Colo320WT。应用1×10^(-8) mol/L 胃泌素以时间梯度0、1、6、12、24、48 h 干预 Colo320WT 细胞,同时应用胃泌素受体拮抗剂 L365,260干预 Colo320WT 细胞30 min,再加1×10^(-8) mol/L 胃泌素干预12 h。免疫印迹检测Colo320WT 细胞中的磷酸化黏着斑激酶(FAK)Tyr397和总 FAK 表达。共聚焦显微镜观察磷酸化FAKTyr397在板状伪足的定位表达。免疫共沉淀检测 FAK-Src-p130^(Cas)-Dock180信号复合物的形成。Pull-down 测定 Rac 活性。结果随着胃泌素干预时间的延长,磷酸化 FAKTyr397的表达量呈增加趋势,且12 h 达最大,但对总的 FAK 无明显影响,磷酸化的 FAKTyr397/FAK 比值分别为2.82%、9.28%、22.62%、38.59%、28.41%、14.94%。L365,260阻断后的磷酸化的 FAKTyr397/FAK 的比值为7.21%。定位在板状伪足上磷酸化的 FAKTyr397也呈增加趋势,12 h 达最大。免疫共沉淀检测发现 FAK、Src、p130^(Cas)和 Dock180在胃泌素的干预下形成了信号复合物。Pull-down 测定证明胃泌素能活化 Rac,但对总 Rac 表达无影响。胃泌素受体拮抗剂 L365,260阻断可胃泌素引起的效应。结论胃泌素能增加大肠癌细胞的侵袭力可能是通过其与受体作用后使 FAK 发生磷酸化,促进磷酸化FAKTyr397定位在板状伪足、使 FAK-Src-p130^(Cas)-Dock180信号复合物形成以及活化 Rac。
Objective To explore the molecular mechanism of increasing the invasion of colon cancer cells by gastrin 17. Methods The plasmid pCR 3. 1/GR expressing the gastrin receptor cholecystokinin-2 receptor （CCK-2R） was transfected into colonic carcinoma cells of the line Colo320 by Lipofectamine2000 . The clones expressing stably CCK-2R were screened by G418 and named as Colo320WT cells. The expression levels of gastrin receptor of the Colo320 and Colo320WT cells were assayed by RT-PCR and Western blotting. The Colo320WT cells were treated by gastrin-17, and the expression levels of phosphorylated FAKTyr397 and total focal adhesion kinase （FAK） in the Colo320WT cells at the time points 0, 1, 6, 12, 24, and 48 h were detected by Western blotting. Another Colo320WT cells were treated by L365,260, gastrin17 receptor blocker, for 30 minutes firstly and then treated by gastrin17 again for 1：2 hours, ancl then Western blotting was used to detect the expression levels of phosphorylatecl FAKTyr397 and total focal adhesion kinase （FAK） at the time points 0, 1, 6, 12, 24, and 48 h . Cofocal microscopy was used to observe the phosphorylated FAKTyr397 localizing in the lamellipoda. The information of FAK-Src-p130^Cas-Dock180 signaling complex was assayed by coimmuniprecipation and immunity blotting. The level of Rac-GTPase was tested by pull clown assay. Results The level of phosphorylatecl FAKTyr397 expression in the Colo320WT cells after the gastrinl7 intervention increased time-dependently ancl peaked at the time point of 12 h,and the phosphorylated FAKTyr397 expression in the Colo320WT cells treated by L365, 260 decreased remarkably, but the level of total FAK remained unchanged. The phosphorylated FAKTyr397/FAK levels were 2. 82%, 9. 28%, 22. 62%, 38. 59%, 28.41%, and 14.94% , O, 1, 6, 12, 24, and 48 h after the gastrin17 treatment respectively , and the level was 7.21% after L365,260 treatment. The amount of phosphorylated FAKTyr397 localizing in the lamellipoda of the Colo320WT cells that were treated by gastrinl7 increased time-dependently and peaked at the time-point 12 h. FAK-Src-p13O^Cas-Dock180 signaling complex was formed in the Colo320WT cells stimulated with gastrinlT. Gastrinl7 activated Rac, but did not affect the total Rac expression. Conclusion The mechanism of increasing the colon cancer cells＇ invasion by gastrinl7 is probably that gastrinl7 makes FAK-Tyr397 phophorylated and be localized to lamellipoda, causes the forming of FAK-Src-p130^Cas-Dockl80 signaling complex when it is bound to its receptor CCK-2, and activation of Rac.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第24期1704-1708,共5页
National Medical Journal of China
基金
国家自然科学基金(30470782)
关键词
结肠肿瘤
肿瘤侵润
伪足
Colonic neoplasmas
Neoplasm invasiveness
Psedopodia
