创伤愈合过程中单核巨噬细胞集落刺激因子调控新生血管化的分子机制

Molecular mechanism of granulocyte/macrophage colony-stimulating factor regulating neovascularization during wound healing

目的观察创伤愈合过程中单核巨噬细胞集落刺激因子（granulocyte／macrophage colony—stimulating factor，GMCSF）经ERK通路活化核因子（nuclear factor，NF）-κB诱导创伤后人皮肤成纤维细胞（human dermal fibroblasts，HDFs）生成血管内皮生长因子（vascular endothelial growth factor，VEGF），并探讨相关机制。方法分离培养创伤部位HDFs，并用GMCSF处理。作用不同时间后，采用RT—PCR和ELISA分别检测HDFs VEGF mRNA和蛋白水平；应用GMCSF作用HDFs后，采用Western blot观察ERK磷酸化水平的改变；进一步应用ERK通路特异性抑制剂PD98059预处理HDFs。再用GMCSF刺激已预处理过的细胞，收集细胞和上清液，采用Western blot检测VEGF蛋白水平的变化；进一步通过抑制ERK信号通路，采用免疫荧光检测NF—κB的活化。另外，应用核质抽提试剂盒分离胞质和胞核，采用Westem blot检测NF—κB的活化。结果随着GMCSF浓度的增加，VEGF mRNA及蛋白水平也逐步增加，呈一定的剂量依赖性；GMCSF作用于HDFs2h后，VEGF mRNA水平开始升高，至4～6h达到峰值。GMCSF能够显著活化ERK磷酸化过程；与GMCSF组相比，ERK信号通路特异性抑制剂PD98059能显著抑制GMCSF诱导VEGF的表达（P〈0．05）。免疫荧光和Western blot结果显示，抑制ERK后NF—κB的活化受到显著抑制。结论GMCSF可通过ERK信号通路活化NF—κB从而诱导HDFs VEGF的表达。

Objective To observe production of vascular endothelial growth factor （VEGF） induced by granulocyte/macrophage colony-stimulating factor （GMCSF） via ERK nerve growth factor （NF） -κB singling pathway in human fibroblasts during wound healing and explore relating mechanism. Methods Human fibroblasts from the injured skin were used for this study and treated with GMCSF. RT-PCR was used for analyzing the protein and mRNA levels of VEGF and Western blotting was employed to determine the phosphorylation of ERK. The fibroblasts were pre-treated with ERK specific inhibitor PD98059 and further treated with GMCSF, then the fibroblasts and the supernatant were collected for detection of protein level of VEGF by means of Western blot. ERK signal pathway was inhibited to detect the activation of NF-κB by means of immunofluorescence staining. Furthermore, the nuclear and cytoplasmic extraction kit was used to separate the cytoplasm and nucleus and Western blot employed for observation of the NF-κB activation. Results The mRNA level and protein level of VEGF were increased significantly with treatment with higher concentration of GMCSF in a dose-dependent manner. VEGF mRNA level was increased two hours after administration with GMCSF and reached peak at 4-6 hours. GMCSF could remarkably activate the ERK phosphorylation. Compared with GMCSF, the ERK specific inhibitor PD98059 inhibited significantly the effect of GMCSF in inducing VEGF expression （ P 〈 0.05 ）. Western blot and immunofluorescence staining analyses showed that the activation of NF-κB was inhibited with reduced production of VEGF after GMCSF treatment. Conclusion GMCSF up-regulates production of VEGF through activating NF-κB via ERK signal pathway in the human fibroblasts.