吡非尼酮对人椎板切除术后硬膜外瘢痕细胞的作用及机制研究

Effects of pirfenidone on epidural scar cells after laminectomy and related mechanisms

目的探讨吡非尼酮对人椎板切除术后硬膜外瘢痕细胞的作用及机制。方法采用体外组织块培养人硬膜外瘢痕细胞并建立细胞系,分别加入不同浓度的吡非尼酮培养,CCK-8法检测细胞活力,细胞划痕法观察细胞迁移,TUNEL法检测细胞凋亡,Western blot分析转化生长因子β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白、Smad2、磷酸化的Smad2(phospho-Smad2)、Smad3、磷酸化的Smad3(phospho-Smad3)表达。结果成功分离培养人硬膜外瘢痕成纤维细胞。吡非尼酮可抑制成纤维细胞的活性(P<0.05),且并未增加成纤维细胞的凋亡(P>0.05)。吡非尼酮可抑制细胞的迁移,并抑制α-SMA、Ⅰ型胶原蛋白表达及细胞增殖相关蛋白的表达和磷酸化。结论吡非尼酮可抑制瘢痕成纤维细胞的活性及迁移,其机制可能是通过抑制TGF-β/Smad通路及肌成纤维细胞转化实现的。

Objective To investigate the effects of pirfenidone on epidural scar cells after laminectomy and related mechanism.Methods The human epidural scar cells were cultured in vitro.Then,the cells were exposed to different concentrations of pirfenidone.Their viability was measured by CCK-8 assay.Their migration was measured by the wound healing assay.The cell apoptosis was detected by in situ terminal transferase labeling(TUNEL).The levels of transfer growth factorβ1(TGF-β1),α-SMA,collagen type I,Smad2,phospho-Smad2,Smad3 and phospho-Smad3 were measured by Western blot.Results Human epidural scar fibroblasts were successfully isolated and cultured.Pirfenidone decreased the viability of fibroblasts(P<0.05),but did not stimnlate increase the apoptosis of fibroblasts(P>0.05).Pirfenidone inhibited cell migration and the expression ofα-SMA,collagen type I,cell proliferation-related proteins and proteins phosphorylation.Conclusions Pirfenidone inhibits the viability and migration of fibroblasts,which may be associated with inhibition of the TGF-β/Smad pathway and myofibroblast transformation.