沙利度胺对组胺活化后人真皮成纤维细胞胶原蛋白表达及其调节因子的影响

Effects of Thalidomide on the Expression of Collagen ⅠA1 and Regulatory Factor in Histamine-activated Human Dermal Fibroblasts

目的 研究沙利度胺对组胺活化后正常人真皮成纤维细胞Ⅰ型胶原蛋白A1 (COLⅠA1)表达及调节因子TGF-β1、MMP-1、TIMP-1 mRNA表达的影响,初步探讨沙利度胺对细胞外相关基质蛋白的抑制作用,以了解其抗真皮纤维化的潜在作用。方法 使用第4代正常人真皮成纤维细胞用于试验。细胞分为空白对照组、沙利度胺组、组胺组及组胺+沙利度胺组,分别用DMEM培养基、10μmol/L沙利度胺、100μmol/L组胺及100μmol/L组胺+10μmol/L沙利度胺处理成纤维细胞,提取各组细胞RNA,荧光定量PCR法检测COLⅠA1及TGF-β1、MMP-1、TIMP-1 mRNA表达,Western-blotting分析COLⅠA1蛋白水平表达。提取各组细胞上清液,用羟脯氨酸比色法检测细胞羟脯氨酸含量。结果 组胺刺激正常人真皮成纤维细胞后,COLⅠA1 mRNA及蛋白表达和羟脯氨酸分泌量较空白对照均升高(P<0.05);沙利度胺单独作用于成纤维细胞后,COLⅠA1 mRNA表达较空白对照降低(P<0.05),而羟脯氨酸分泌量差异无统计学意义(P>0.05);沙利度胺作用于组胺刺激后的成纤维细胞、COLⅠA1 mRNA的表达及羟脯氨酸分泌量较前者(未经组胺处理组)显著降低,且沙利度胺对组胺刺激后的成纤维细胞COLⅠA1mRNA表达抑制率(85.55±3.07)%高于未用组胺处理的抑制率(35.25±2.01)%。TGF-β1、MMP-1、TIMP-1 mRNA表达的结果显示:与空白对照组比较,组胺处理组TGF-β1、MMP-1 mRNA表达升高(P<0.05),但TIMP-1 mRNA表达差异无统计学意义(P>0.05);沙利度胺组较空白对照组比较TGF-β1、TIMP-1 mRNA表达降低,MMP-1 mRNA表达升高(P<0.05);而沙利度胺作用于经组胺刺激后的成纤维细胞TGF-β1、TIMP-1 mRNA较组胺组降低,MMP-1 mRNA表达显著升高(P<0.05)。结论 10μmol/L沙利度胺能抑制经组胺活化后COLⅠA1 mRNA及蛋白水平的表达和羟脯氨酸的分泌量,同时抑制胶原蛋白产生的正向调节因子TGF-β1、TIMP-1 mRNA表达、促进负向调节因子MMP-1表达。

Objective To explore the effects of thalidomide on the expressions for COLⅠA1, TGF-β1, MMP-1 and TIMP-1 mRNA in human dermal fibroblasts activated by histamine. To investigate the possible inhibitory effects of thalidomide, on the expression of functional extracellular matrix proteins in both of the control and Histamine treated fibroblast, in order to reveal the potential anti-fibrotic properties of thalidomide.Methods The fourth passages of human dermal fibroblasts were selected for experiments. Cells were treated with 10 μmol/L of thalidomide, 100 μmol/L of histamine, and both those reagents in the culture flask. The control group was treated with the medium.PCR was used to detect the mRNA expression of COLⅠA1,TGF-β1,MMP-1 and TIMP-1. The secretion mount of hydroxyproline, the expression of COLⅠA1 were detected by hydroxyproline assay and Western blotting.Results The results showed that the expressions of COLⅠA1 on the mRNA level, protein level and the secretion of hydroxyproline all increased after stimulation of human normal dermal fibroblasts by histamine(P<0.05).The expression of COLⅠA1 mRNA was inhibited(P<0.05), but the hydroxyproline was not affected by thalidomide(P>0.05) in normal fibroblasts. The expression of COLⅠA1 mRNA and secretion mount of hydroxyproline could be inhibited significantly after activated by histamine. Furthermore, in comparing to no-histamine treated group the inhibition ratios of COLⅠA1 mRNA significantly decreased(85.55±3.07)% vs.(35.25±2.01)%;after activated by histamine, the expressions of TGF-β1, MMP-1 mRNA increased(P<0.05) and TIMP-1 mRNA was not affected(P>0.05) in histamine treated group, but in thalidomide-treated group showed the opposite results. Moreover, thalidomide showed significant effects of in histamine treated group, significantly decreased expressions of TGF-β1,TIMP-1 mRNA and increased expressions of MMP-1 mRNA(P<0.05).Conclusion 10 μmol/L thalidomide showed a positive effect on inhibition of COLⅠA1 mRNA expression and concentration of hydroxyproline. Meanwhile it demonstrated an downregulating effect on positive factors TGF-β1,TIMP-1 mRNA and upregulating effect on negative factors MMP-1 for the production of collagen in histamine-activated human dermal fibroblasts. Therefore thalidomide may play a potential role in the treatment of hypertrophic scar..