TGF-β1通过上调Shh信号诱导大鼠脑膜成纤维细胞向肌成纤维细胞转化

Transforming growth factor-β1 induces transformation of rat meningeal fibroblasts into myofibroblasts by upregulating Shh signaling

目的探讨TGF-β1在诱导脑膜成纤维细胞转化为肌成纤维细胞中对Shh信号的影响。方法新生24 h SD大鼠脑膜原代成纤维细胞经Ⅳ型胶原酶提取纯化。原代脑膜成纤维细胞随机分为3个组:分别为空白对照组、10 ng/mL TGF-β1处理组(TGF-β1组)、10 ng/mL TGF-β1联合20μmol/L TGF-β1受体抑制剂SB-431542组(TGF-β1+SB组)。各组细胞在药物处理前均用无血清培养基培养24 h后换用加入了药物的DMEM/F12完全培养基处理72 h。采用CCK-8法检测各实验组成纤维细胞的增殖能力;细胞划痕实验检测各实验组成纤维细胞的迁移能力;免疫荧光法检测各实验组纤维连接蛋白(Fn)的表达;Westernblotting检测各实验组Fn、α-平滑肌肌动蛋白(α-SMA)、Shh蛋白的表达;q-PCR检测各实验组细胞Shh mRNA的表达。结果TGF-β1可促进原代脑膜成纤维细胞的增殖(P<0.05);TGF-β1可促进原代脑膜成纤维细胞的迁移(P<0.05)。TGF-β1可促进成纤维细胞向肌成纤维细胞的转化并增加Fn的分泌(P<0.05)。TGF-β1处理后可上调Shh蛋白和mRNA的表达(P<0.05),TGF-β1受体抑制剂SB-431542则可抑制TGF-β1引起的原代脑膜成纤维细胞向肌成纤维细胞的转化并减少纤维连接蛋白的分泌(P<0.05)。结论TGF-β1可通过上调Sonic Hedgehog信号通路中Shh蛋白的表达,诱导脑膜成纤维细胞转化为肌成纤维细胞。

Objective To investigate the effect of TGF-β1 on Shh signaling pathway during the transformation of meningeal fibroblasts into myofibroblasts.Methods Primary meningeal fibroblasts were isolated from neonatal(24 h)SD rats and purified using typeⅣcollagenase.The isolated cells were treated with 10 ng/mL TGF-β1 alone or in combination with 20μmol/L SB-431542(a TGF-β1 receptor inhibitor)for 72 h,and the changes in proliferation and migration abilities of the fibroblasts were assessed with CCK-8 assay and cell scratch test.The expression of fibronectin(Fn)was detected with immunofluorescence assay,and Western blotting was performed to examine the expressions of Fn,α-SMA and Shh protein in the cells;the expression of Shh mRNA was detected with real-time fluorescence quantitative PCR.Results TGF-β1 treatment obviously enhanced the proliferation and migration of primary meningeal fibroblasts(P<0.05),and promoted the transformation of meningeal fibroblasts into myofibroblasts and the secretion of Fn(P<0.05).TGF-β1 treatment also upregulated the expression of Shh at both protein and mRNA levels(P<0.05).Treatment with SB-431542 partially blocked the effect of TGF-β1 on the transformation of meningeal fibroblasts(P<0.05).Conclusion TGF-β1 can induce the transformation of meningeal fibroblasts into myofibroblasts by up-regulating Shh expression in Sonic Hedgehog signaling pathway.