目的:探讨N-乙酰基-丝氨酰-天门冬氨酰-赖氨酰-脯氨酸(Ac-SDKP)是否通过对转化生长因子-β1(TGF-β1)介导的ROCK/SRF/α-SMA信号转导通路活化的调节,阻抑肺泡Ⅱ型上皮细胞向间质细胞转化,即上皮-间质转化(EMT),进而发挥其抗(矽)肺纤维化的作用.方法:培养人A549肺泡Ⅱ型上皮细胞株,免疫细胞化学显色检测角蛋白、波形蛋白及α-平滑肌肌动蛋白(α-SMA)的表达；免疫印迹检测E-钙黏蛋白(E-cad)、波形蛋白及Rho相关卷曲蛋白激酶(ROCK)、血清反应因子(SRF)、α-SMA及Ⅰ型、Ⅲ型胶原蛋白表达；Real-Time PCR检测ROCK、SRF、α-SMA的表达情况.结果:在TGF-β1的诱导下,上皮细胞向肌成纤维细胞的形态改变,伴随着上皮标志物角蛋白、E-cad降低,而间质细胞标志物波形蛋白增高,α-SMA表达并增多.与对照组相比,TGF-β1诱导刺激组ROCK、SRF、α-SMA蛋白和基因表达水平上调,Ⅰ型、Ⅲ型胶原蛋白表达上调.给予ROCK阻滞剂Y-27632、Ac-SDKP干预后,ROCK、SRF、α-SMA及Ⅰ型、Ⅲ型胶原表达显著降低.结论:Ac-SDKP通过阻断TGF-β1介导的ROCK/SRF/α-SMA信号转导通路,阻抑肺泡Ⅱ型上皮细胞向肌成纤维细胞的转化及胶原的合成,进而发挥其抗(矽)肺纤维化的作用.%Objective:To investigate whether Ac-SDKP can regulate the ROCK/SRF/-SMA pathway mediated by TGF-β1 on EMT and play the role of anti-(Si) pulmonary fibrosis.Methods:Human type Ⅱ alveolar epithelial cells (A549) were used in the experiment.The evidence of EMT was assessed by morphology under a phase-contrast microscope.The expressions of the phenotype markers of the epithelial cells cytokeratin and mesenchymal marker vimentin,α-SMA were identified by immunocytochemistry.The expressions of E-cad,vimentin,ROCK,SRF α-SMA protein and type Ⅰ,type Ⅲ collagen were measured by Western blotting.ROCK,SRF a-SMA were also evaluated by measuring mRNA level using real-time PCR.Results:The process of EMT induced by TGF-β1 was accompanied by morphological alteration and the expression of the fibroblast phenotypic marker vimentin and myofibroblast phenotypic marker α-SMA,concomitant with a down-regulation of the epithelial phenotypic markers E-cad and cytokeratin.Compared with the control group,the expressions of ROCK,SRF,α-SMA on the level of both protein expression and gene detection increased significantly after the stimulation of TGF-β1.Collagen Ⅰ and Collagen Ⅲ were up-regulated too.While they were decreased significantly in Y-27632 (ROCK blockers) treating group and Ac-SDKP group.Conclusion:Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Rho/ROCK pathway activation.Ac-SDKP,similar to Y-27632,can inhibit the process of differentiation from epithelial cells to myofibroblasts by blocking TGF-β1 which induces ROCK/SRF/α-SMA signal transduction pathway,decreases the expressions of type Ⅰ and type Ⅲ collagen and enhances its anti-silicotic effects.
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