FIP200对FN诱导的人类呼吸道上皮细胞增殖的影响

目的研究 FIP200(FAK-family interacting protein of 200 kDa)对细胞外基质成份诱导的呼吸道上皮细胞增殖和细胞周期演进的影响及其作用机制.方法通过纤连接蛋白(FN)诱导培养呼吸道上皮细胞增殖,以FIP200反义寡核苷酸(ODN)经脂质体转染细胞;利用四唑盐(MTT)比色实验研究呼吸道上皮细胞增殖情况;采用流式细胞术分析呼吸道上皮细胞细胞周期各期分布情况;以Western blot检测FIP200和FAK蛋白表达水平;以免疫共沉淀检测FIP200和FAK结合情况和FAK磷酸化程度.结果 FN诱导的呼吸道上皮细胞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,FIP200表达有降低趋势,FAK与FIP200的结合程度显著减低;与40mg/L FN组相比,经脂质体转染了反义FIP200寡核苷酸的气道上皮细胞MTT吸光度值显著增高,G1期细胞数明显减少,S期和G2期细胞数显著增多,FIP200表达水平显著降低,FAK表达水平无明显变化,与FIP200结合的FAK显著降低, FAK酪氨酸磷酸化程度明显增高.结论内源性FIP200和FAK的结合抑制FAK的活化,从而抑制呼吸道上皮细胞增殖和细胞周期演进,内源性FIP200作为FAK的抑制剂而存在;FN能够促使FAK和FIP200结合的解离而活化FAK,从而促进细胞增殖.

EFFECT OF FIP200 ON PROLIFERATION OF HUMAN RESPIRATORY WAY EPITHELIAL CELLS STIMULATED BY FIBRONECTIN

Objective To study the effects of FIP200 on the cells proliferation and cell cycle of human respiratory way epithelial cells stimulated by fibronectin.Methods Cultured human respiratory way epithelial cells were stimulated with fibronectin. FIP200 antisense oligodeoxynucleotides(ODNs)were transfected into human respiratory epithelial cells by the actionic lipid method. The cell proliferation was measured with MTT and the distribution of different cell cycles with flow cytometry; FIP200 and FAK expression levels were analyzed by Western blot;the association of FIP200 with FAK and the degree of tyrosine phosphorylation of FAK was analyzed by immunoprecipitation.Results FN enhanced the proliferation of human respiratory way epithelial cells and decreased dramatically the number of epithelial cells in G_ 1 phase of the cell cycle but increased the number in S and G_ 2 phases. The expression level of FAK and the phosphorylation of FAK were increased remarkably. FIP200 antisense ODNs improved cell proliferation, decreased respiratory epithelial cells in G_ 1 phase and increased the the cells in S and G_ 2 phases. Meanwhile, the expression of FIP200 was inhibited drastically, the expression of FAK was normal, but the association of FIP200 with FAK was decreased significantly and the phosphorylation of FAK was increased drastically.Conclusion The association of endogenous FIP200 with FAK can inhibit the activation of FAK, by which FIP200 can inhibit the proliferation of the epithelial cells and the progression of the cell cycle, so endogenous FIP200 functions as a protein inhibitor for FAK kinase activity. FN can disrupt the association of FIP200 with FAK, which can activate FAK and improve the repair process of the respiratory way epithelia.