CAJ | 학술논문

目的观察创伤愈合过程中单核巨噬细胞集落刺激因子(granuiocyte/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Western 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HDFs 2 h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HDFsVEGF的表达。
목적 관찰창상유합과정중단핵거서세포집락자격인자(granuiocyte/macrophage colony - stimulating factor,GMCSF)경ERK통로활화핵인자(nuclear factor，NF) - Κb유도창상후인피부성섬유세포（human dermal fibroblasts,HDFs）생성혈관내피생장인자(vascular endothelial growth factor，VEGF)，병탐토상관궤제。방법 분리배양창상부위HDFs，병용GMCSF처리。작용불동시간후，채용RT - PCR화ELISA분별검측HDFs VEGF Mrna화단백수평；응용GMCSF작용HDFs후，채용Western blot관찰ERK린산화수평적개변；진일보응용ERK통로특이성억제제PD98059예처리HDFs。재용GMCSF자격이예처리과적세포，수집세포화상청액，채용Western blot검측VEGF단백수평적변화；진일보통과억제ERK신호통로，채용면역형광검측NF - Κb적활화。령외，응용핵질추제시제합분리포질화포핵，채용Western blot검측NF - Κb적활화。결과 수착GMCSF농도적증가，VEGF Mrna급단백수평야축보증가，정일정적제량의뢰성；GMCSF작용우HDFs 2 h후，VEGF Mrna수평개시승고，지4～6h체도봉치。GMCSF능구현저활화ERK린산화과정；여GMCSF조상비，ERK신호통로특이성억제제PD98059능현저억제GMCSF유도VEGF적표체(P＜0.05)。면역형광화Western blot결과현시，억제ERK후NF - Κb적활화수도현저억제。결론 GMCSF가통과ERK신호통로활화NF - Κb종이유도HDFsVEGF적표체。
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 PD98059inhibited 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.