丝裂原活化蛋白激酶通路对成纤维细胞内游离钙的影响

EFFECTS OF MITOGEN-ACTIVATED PROTEIN KINASE ON FREE CALCIUM IN CULTURED HUMAN FIBROBLAST

为观察碱性成纤维细胞生长因子(bFGF)对离体热烫伤后成纤维细胞内钙离子的作用以及丝裂原活化蛋白激酶 (MAPK)通路对胞内游离钙的影响 ,将培养的人成纤维细胞进行热损伤刺激后分成 4组 :①bFGF处理组 (10ng/ml) ;②预先加入PD980 5 9(10 μmol/L)阻断剂 30min,再行bFGF(10ng/ml)刺激 ;③预先加入SB2 0 35 80 (10 μmol/L)阻断剂 30min ,再行bFGF刺激 (10ng/ml) ;④同时加入PD980 5 9(10 μmol/L)和SB2 0 35 80 (10 μmol/L) 2种阻断剂 30min,再加入bFGF(10ng/ml)。应用特异性Ca2 + 荧光指示剂Fluo 3/AM负载细胞 ,激光共聚焦显微镜检测细胞内游离钙的浓度。结果显示 ,受热刺激的成纤维细胞荧光强度较弱。加入bFGF后可促使成纤维细胞中游离Ca2 +浓度升高 ,分别预先加入PD980 5 9和SB2 0 35 80拮抗剂的成纤维细胞 ,再加入bFGF ,胞内钙离子浓度出现不同的钙振荡现象。同时加入 2种阻断剂后胞内钙离子浓度则迅速降低。表明bFGF引起热损伤后的成纤维细胞中游离Ca2 + 浓度的增加 。

To investigate the effects of basic fibroblast growth factor (bFGF) on intracellular free Ca 2+ of heating injury, and observe the relationship between mitogen activated protein kinases(MAPKs)signal pathways and Ca 2+ mobilization. Cultured human fibroblasts were heated at 45°C for 10min, then divided into four groups :①basic fibroblast growth factor (10ng/ml) treatment; ②preincubated cells with PD98059 (10μmol/L) for 30 min followed by bFGF (10ng/ml); ③preincubated cells with SB203580 (10μmol/L) for 30min followed by bFGF (10ng/ml); ④preincubated cells with PD98059 (10μmol/L) and SB203580 (10μmol/L) for 30min followed by bFGF (10ng/ml). The cells were incubated with fluorescence Ca 2+ dye fluo 3/AM at 37 C for 30min, then measured by using laser scanning confocal microscope. The results showed fluorescent intensity of fibroblast heating injury was weak, the concentration of intracellular free Ca 2+ increased after stimulation with bFGF treatment. PD98059 and SB203580 could induce calcium oscillation. A rapid decrease of fluorescent intensity was observed after cells were preincubated with PD98059 and SB203580 at the same time. bFGF induced an increase of cytoplasmic and intracellular free Ca 2+ concentrations. It is suggested that MAPKs signaling pathway has a feedback regulation for free Ca 2+ mobilization.