粘着斑激酶活性对气道上皮细胞增殖的影响

Effects of Focal Adhesion Kinase Phosphorylation on Proliferation of Human Airway Epithelial Cell Stimulated by Fibronectin

目的:研究粘着斑激酶(focal adhesion kinase,FAK)磷酸化对细胞外基质成份诱导的气道上皮细胞增殖和细胞周期演进的影响,探讨粘着斑激酶活化对气道上皮细胞损伤后修复影响的机制。方法:通过纤维连接蛋白(finbronectin,FN)诱导培养的气道上皮细胞,利用四唑盐(MTT)比色实验研究气道上皮细胞增殖情况,采用流式细胞术分析气道上皮细胞细胞周期各期分布,以Western blot和免疫共沉淀检测FAK表达水平及其酪氨酸磷酸化程度;以FAK反义寡核苷酸(ODNs)经脂质体转染细胞,观察其对FAK磷酸化、细胞增殖和细胞在细胞周期各期分布的影响。结果:FN诱导的气道上皮细胞MTT吸光度明显增强,G1期细胞数量明显减少,S期和G2期细胞数量明显增多,同时FAK表达水平及其酪氨酸磷酸化程度显著增高;经脂质体转染了反义FAK寡核苷酸的气道上皮细胞MTT吸光度值显著降低,G1期细胞数明显增多,S期和G2期细胞数显著减少,FAK表达水平及其酪氨酸磷酸化程度降低,且与G1期细胞数量的增多成显著负相关,与S期、G2期细胞数量和MTT吸光度的减少成明显正相关。结论:FAK是细胞外基质诱导气道上皮细胞增殖的重要信号分子,其活化促进气道上皮细胞的增殖和细胞周期的演进,在气道上皮细胞损伤后修复过程中起重要作用。

Objective： To investigate the effects of FAK phosphorylation on the cell proliferation and cell cycle of human airway epithelial cell stimulated by fibronectin. Methods. The cultured human airway epithelial cells were stimulated by fibronectin. The proliferation and distribution of cellular cycles of the cells were measured with MTT and flow cytometry, respectively; FAK expression level was analyzed by western blot and the degree of Tyrosine phosphorylation was by immunoprecipitation. FAK antisense oligodeoxynucleotides （ODNs）were transfected into human airway epithelial cells by actionic lipid method. Results： FN improved the proliferation of human airway epithelial eell and decreased dramatically the number of epithelial cells in phase G1, of cell cycle and increased the number of that in phases S and G2 ; The expression level and pho-sphorylation of FAK were increased magnificently. FAK antisense ODNs inhibited cell proliferation,arrested airway epithelial cells in phase G, and decreased the number of phases S and G2 of cell cycle; meanwhile the expression and phosphorylation of FAK were inhibited drastically. The latter changes were positively correlated to the increased number of epithelial cells in phase G, ,and negatively correlated to the proliferation and the number of epithelial cells in phases S and G2. Conclusion. FAK phosphorylation and FAK-mediated signal transduction may play important roles in the proliferation and progression of cell cycle of human epithelial cells,so activation of FAK may involved in the repair wound-process of airway epi-thelial.