沙利度胺对HaCaT细胞分泌血管内皮细胞生长因子及肿瘤坏死因子α的影响

Effects of thalidomide on the expression and secretion of vascular endothelial growth factor and tumor necrosis factor-alpha by a human keratinocyte cell line HaCaT

目的探讨沙利度胺对HaCaT细胞增殖及血管内皮细胞生长因子（VEGF）和肿瘤坏死因子α（TNF—α）表达的影响。方法体外培养的HaCaT细胞分为阴性对照组及不同浓度沙利度胺实验组；采用水溶性四氮唑法（WST-1）检测沙利度胺对HaCaT细胞增殖的影响，实时定量PCR法检测HaCaT细胞VEGF及TNF-αmRNA的表达水平，ELISA法检测HaCaT细胞VEGF及TNF—α蛋白的表达水平。采用单因素方差分析进行统计分析。结果沙利度胺浓度在0．01—100nmol／L之间时，可抑制VEGFmRNA（0．439～0．634，P〈0．01）与蛋白（0．587～0．923，P〈0．05）的表达；浓度在0．1～100nmol／L之间时，可抑制TNF—αmRNA（0．493～0．587，P〈0．05）与蛋白（0．408～0．617，P〈0．01）的表达。结论沙利度胺在一定浓度范围可抑制角质形成细胞增殖及减少VEGF及TNF-α表达。

Objective To investigate the effect of thalidomide on the proliferation of as well as the expression and secretion of vascular endothelial growth factor （VEGF） and tumor necrosis factor-alpha （TNF-α） by the human keratinocyte cell line HaCaT. Methods Cultured HaCaT cells were divided into several groups to be treated with dimethyl sulfoxide （negative control group） and various concentrations （0.01nmol/L - 100 μmol/L） of thalidomide （experimental groups） respectively for 20 to 24 hours. Subsequently, water soluble tetrazolium-1 （WST-1） assay was performed to estimate cellular proliferation, real time quantitative PCR to detect the mRNA expression of VEGF and TNF-α in HaCaT cells, and enzyme-linked immunosorbent assay （ELISA） to quantify the protein expressions of VEGF and TNF-α in the culture supernatants of HaCaT cells. Statistical analysis was done by one-way analysis of variance with least significant difference post hoc test. Results The survival rate of HaCaT cells was 74.3%, 82.9% and 90.8% after 24-hour treatment with thalidomide of 100, 10 and 1 μmol/L respectively, significantly lower than that in the negative control group （100%, all P 〈 0.01）. A significant decrease was induced in the mRNA expression （0.439- to 0.634-fold change, all P 〈 0.01 ） and supernatant level （（0.587- to 0.923-fold change, P 〈 0.05） of VEGF in HaCaT cells by thalidomide of 0.01 - 100 nmol/L, as well as in the mRNA expression （0.493- to 0.587-fold change, P 〈 0.05） and supernatant level （0.408- to 0.617-fold change, P 〈 0.01） of TNF-α by thalidomide of 0.1 - 100 nmol/L. Conclusion Within a certain range of concentration, thalidomide could suppress the proliferation of, as well as the expression and secretion of VEGF and TNF-α by, HaCaT cells.