TNF-α活化人瘢痕疙瘩成纤维细胞NF-κB的实验研究

目的：观察肿瘤坏死因子－α（TNF-α）对瘢痕疙瘩成纤维细胞（KFB）以及正常皮肤成纤维细胞（NSF）核因子-kappa B（NF-κB）的影响，以探讨瘢痕疙瘩的发生机制。 方法:原代培养成纤维细胞；免疫荧光技术观察NF-κB p65和IκB-α在静息状态和TNF-α刺激后的成纤维细胞中分布；应用TransAMTM NF-κB p65 kit试剂盒检测NF-κB p65 DNA结合活性；应用Western blotting检测IκB-α蛋白水平。 结果: TNF-α刺激后，NF-κB p65从细胞浆转移至细胞核；NF-κB p65 DNA结合活性水平在刺激后1 h达到高峰，4 h接近正常；细胞浆IκB-α蛋白水平在刺激后15 min降至最低值，4 h基本接近正常；KFB较NSF对TNF-α的刺激更为敏感。结论: KFB较NSF对TNF-α活化 NF-κB更为敏感, 可能是瘢痕疙瘩形成的潜在发病机制。

NF-κB activation in keloid fibroblasts stimulated with TNF-α

AIM: To investigate NF-κB p65 activation and IκB-α expression in keloid fibroblasts (KFB) and normal skin fibroblasts (NSF) stimulated with TNF-α and to explore the underlying molecular pathogenesis of keloid formation. METHODS: Primary KFB was cultured. The location of NF-κB p65 and IκB-α in KFB and NSF at quiescent condition and the nuclear translocation of NF-κB p65 after TNF-α stimulation were observed by immunofluorescence technique. NF-κB p65 DNA binding activity was detected with TransAMTM NF-κB p65 kit. The IκB-α protein level was determined by means of Western blotting technique. RESULTS: After stimulated with TNF-α, NF-κB p65 translocated into the nucleus. NF-κB p65 DNA binding activity increased to its maximum at 1 h and was dropped to normal at 4 h. TNF-α induced most degradation of IκB-α at 15 min and became detectable in cytoplasm after 4 h. KFB showed more sensitive ability to TNF-α stimulation than NSF. CONCLUSION: NF-κB may play a role in keloid pathogenesis.