Smad7对转化生长因子β1诱导的肺泡上皮细胞向间质细胞转变的影响

Effect of Smad7 on transforming growth factorial-induced alveolar epithelial to mesenchymal transition

目的检测转化生长因子(TGF)β1能否在体外诱导肺泡Ⅱ型上皮细胞向间质细胞转变(EMT),以及 Smad7基因转染能否阻止此种转变及相关信号转导机制。方法采用脂质体法转染Smad7基因到大鼠肺泡Ⅱ型上皮(RLE-6TN);实时荧光 PCR 及免疫印迹法检测转染前后上皮细胞标志物(E-cadherin,CK19)、间质细胞标志物(FN、Vimentin及α-SMA)及 Smad 信号通路相关蛋白表达变化;相差显微镜观察 TGFβ1诱导的肺泡上皮细胞形态改变,其超微结构改变采用透射电镜观察。结果成功转染 Smad7到 RLE-6TN;转染前,在 TGFβ1作用下,RLE-6TN 间质标志物的表达在 mRNA和蛋白水平上调,而上皮标志物表达下调;转染后,其间质标志物下调的同时上皮标志物上调;转染前RLE-6TN 在 TGFβ1作用下 p-Smad2/3表达上调,转染后其表达无明显改变;形态学上,TGFβ1能诱导肺泡上皮发生 EMT,Smad7转染则能阻止 EMT;超微结构上,TGFβ1能诱导肺泡上皮特有板层小体变性、肿胀并随其时间延长最终完全消失。结论 TGFβ1能在体外诱导肺泡Ⅱ型上皮向间质细胞转变,其机制部分与 Smad 信号转导途径相关,Smad7转染能在一定程度上阻止该转变。

Objective To investigate whether transforming growth factorβ1 （TGF-β1） can induce in vitro alveolar epithelial-mesenchymal transition （EMT）, and whether Smad7 gene transfer can block this transition and the possible signaling mechanisnL Methods Rat alveolar type Ⅱ epithelial cells of the line RLE-6TN were cultured. TGFβ1 （3 ng/mL）was added into the culture fluid. Lipofectamine 2000 was used to transfect Smad7 gene to the RLE-6TN cells. The expression of the markers of the epithelial cells, including E-cadherin and cytokeratin-19 （ CK19）, and markers of mesenchymal cells, including fibronection （FN）, vimentin, and α-smooth muscle actin （α-SMA） were assayed using Western blotting and real-time PCIL The morphological alterations were examined by phase-contrast microscope while the ultrastructure changes were examined by electron microscope. Results Smad7 was successfully transfected to the RLE- 6TN cells. Before transfection TGF-β1 treatment could lead to the expression upregulation of the meshenchymal markers and downregulation of the epithelial markers at the levels of both mRNA and protein, and after transfection, the meshenchymal makers were downregulated while the epithelial markers were upregulated. Before the transfection, TGF-β1 treatment could lead to the expression upregulation of phesphorylated Smad2/3 which did not obvious change following transfection. TGFβ1 treatment could induce the EMT process of the RLE-6TN cells. Smad7 gene transfected into the RLE-6TN cells could block the process of EMT. Exposed of the RLE-6TN cells to TGFβ1 resulted in degeneration, tumefaction, and gradual disappearance of the esmiophilic multilamellar bodies, markers of type Ⅱ alveolar epithelial cells. Conclusion Under TGFβ1 treatment, RLE-6TN undergoes a conversion process into myofibroblasts in vitro with the conversion mechanism related to Smad signaling pathway, and transfection of Smad7 gene can partly reverse this process.