纤维连接蛋白对人结肠癌细胞侵袭力的影响

目的研究纤维连接蛋白（fibronectin，FN）对人结肠癌细胞侵袭力的影响，并探讨其中的信号传导机制。方法以递增浓度的FN刺激结肠癌细胞株Colo320，以免疫沉淀和蛋白质印迹法检测结肠癌细胞内黏着斑激酶（focal adhesion kinase，FAK）第397位酪氨酸（tyr-397）磷酸化的状况．以改良Boyden小室法检测相应的细胞侵袭力变化。设计反义寡核苷酸阻断FAK蛋白质表达，再次观察接受FN刺激后．结肠癌细胞内FAK tyr-397磷酸化状况及细胞侵袭力的改变。结果FN能够促进Colo320 FAK tyr-397磷酸化，在一定范围内具有剂量依赖性。FN浓度达10nmol／L时．此作用最显著，继续增加FN浓度．FAK tyr-397磷酸化不再呈现继续增强趋势．当浓度达到100nmol／L时，磷酸化程度反而有所下降；FN可以增强细胞侵袭力，同样在一定范围内具有剂量依赖性；反义寡核苷酸干预后．结肠癌细胞内FAK tyr-397磷酸化及细胞侵袭力均有显著降低（P〈0.01）。结论FN可以有效增强结肠癌细胞的侵袭力，其作用是通过FN—FAK信号通路实现的，FAK的活性形式是其酪氨酸位点的磷酸化．阻断FAK的表达町以减弱FN促进细胞侵袭的作用。

Effect of fibronectin on invasion of colon cancer cell

Objective The investigate the effect of fibronectin on invasion of human colon cancer cells,and on the tyrosine phosphorylation of focal adhesion kinase(FAK).Methods A colorectal cancer cell line,Colo320,was stimulated by fibronectin with increasing doses (0.0.1 nmol/L.1 nmol/L, 10 nmol/L,100 nmol/L).Phosphorylation of FAK tyrosine 397 (tyr-397),was determined by immunoprecipitation and Western-blotting.The invasion of Colo320 was detected by Boyden chamber assay.Moreover.phosphorylation of FAK tyr-397 interfered by antisense oligonucleotides was examined by immunoprecipitation and Western-blotting before and after being stimulated hy fibronectin.Results Invasion and phosphorylation of FAK in Colo320 increased with fibronectin stimulation in dose-depend- ence manner,and reached to climax when concentration of fibronectin approached to 10 nmol/L.But no further increasing was found when the concentration of fibronectin was 100 nmol/L.Compared with the controls,antisense oligonucleotides could decrease phosphorylation of FAK tyr-397 and invasion of the tumor cells significantly (P<0.01).Conclusions Fibronectin can promote invasion of Colo320 cells. FAK plays an important role in this process.Inhibition of FAK expression can decrease colon cancer cell invasion.