地西他滨抑制瘢痕疙瘩成纤维细胞增殖的实验研究

Experimental study on inhibition of proliferation of keloid fibroblasts by decitabine

目的探讨地西他滨对瘢痕疙瘩成纤维细胞增殖及TGF-β/Smad信号通路相关蛋白表达的影响。方法 CCK8法检测不同浓度地西他滨对瘢痕疙瘩成纤维细胞增殖的影响。Western blotting检测药物作用前后瘢痕疙瘩成纤维细胞中转化生长因子β1(TGF-β1)、磷酸化Smad2/3(p-Smad2/3)、Smad4、Ⅰ型胶原α1链(COL1A1)和Ⅲ型胶原α1链(COL3A1)的表达;采用免疫共沉淀法及Smad4 siRNA转染研究p-Smad2/3-Smad4共聚体在瘢痕疙瘩形成中的作用,Real-time PCR检测下游转录因子的表达情况。结果 CCK8法检测结果显示:干预后48 h,随着地西他滨浓度的升高,成纤维细胞生长抑制率总体呈上升趋势,实验选择半数抑制浓度4.38μmol/L作为地西他滨对耳垂瘢痕疙瘩成纤维细胞的干预浓度。药物干预后,瘢痕疙瘩成纤维细胞TGF-β1、p-Smad2/3、COL1A1和COL3A1表达及p-Smad2/3-Smad4共聚体形成显著减少。Smad4被沉默后,瘢痕疙瘩成纤维细胞COL1A1表达减少,转录因子c-jun、runx2、irf-7 mRNA的表达下调。结论地西他滨抑制瘢痕疙瘩成纤维细胞的增殖,使胶原蛋白合成受限,其机制可能与抑制Smad2/3的磷酸化及p-Smad2/3-Smad4蛋白复合体的形成有关。

Objective To evaluate the effects of decitabine on the proliferation of keloid fibroblasts and the expression of proteins relevant to TGF-β / Smad signal pathways. Methods CCK8 assay was adopted to detect the effects of decitabine of different concentrations on the proliferation of keloid fibroblasts. The expressions of transforming growth factor-β1（ TGF-β1）, phosphorylated p-Smad2 /3（ p-Smad2 /3）, Smad4, collagen type I alpha 1（ COL1A1）, and COL3A1 of keloid fibroblasts before and after being intervened by decitabine were detected by the Western blotting. Immunoprecipitation assay and Smad4 siRNA transfection were adopted to explore the role of pSmad2 /3-Smad4 complex during the formation of keloid. Real-time PCR was used to detect the expression of downstream transcriptional factors. Results The results of CCK8 assay showed that the growth inhibition rate of fibroblasts increased with the concentration of decitabine after being intervened for 48 h. IC50 concentration of decitabine of 4. 38 μmol / L was selected as the intervention concentration of ear lobe keloid fibroblasts. After being intervened by decitabine, expressions of TGF-β1, p-Smad2 /3, COL1A1, and COL3A1 of keloid fibroblasts and the formation of p-Smad2 /3-Smad4 complex significantly decreased. The expression of COL1A1 of keloid fibroblasts decreased and mRNA expressions of transcriptional factors c-jun, runx2, and irf-7 were down-regulated after Smad4 was silenced. Conclusion Decitabine can inhibit the proliferation of keloid fibroblasts and the synthesis of collagens. The mechanism may be relevant to the inhibition of phosphorylation of Smad2 /3 and the formation of pSmad2 /3-Smad4 protein complex.