Basic research on the control treatment of pulmonary fibrosis by adenoviral vector which promotes selective gene expression

间充质细胞如成纤维细胞和肌成纤维细胞是肺纤维形成过程中主要的基质生成细胞。TGF β信号在刺激细胞外基质的主要成分I型胶原的表达中起重要作用。最近，tgf - β超家族成员骨形态发生蛋白(BMP)-7被报道在纤维形成中抑制tgf - β1。在这里，我们研究了BMP-7基因转移对肺肌成纤维细胞系成纤维反应的影响，用荧光素酶法检测胶原启动子活性。为此，我们首先从含有胶原蛋白1A2上游序列的转基因小鼠肺组织中建立细胞系。这些细胞系呈梭形，表达vimentin和aSMA，但不表达E-cadherin，这表明它们来源于肺肌成纤维细胞。它们显示出荧光素酶活性依赖于tgf - β1刺激的剂量。该活性也因Smad3过表达而上调，因Smad7过表达而下调。这些细胞系用于评估BMP-7的效果，以供后续检查。在免疫细胞化学检查中，BMP-7 （Ad-BMP-7）的异位表达导致磷酸化smadl /5/8的核定位，抑制Smad3的核定位。Ad-BMP-7降低胶原1A2启动子依赖性荧光素酶活性。Real-time RT PCR结果显示，Ad-BMP-7抑制胶原蛋白1A2、组织金属蛋白酶抑制剂（tissue inhibitor of metalloproteinase）的表达，增加分化抑制剂（inhibitor of differentiation, Id） 2和3的表达，从而抑制tgf - β信号传导。此外，Id2和ID的异位表达降低了肺肌成纤维细胞中胶原1A2启动子依赖性荧光素酶的活性。总之，这些数据表明BMP-7通过在小鼠肺肌成纤维细胞系中表达1d2来拮抗tgf β依赖性纤维生成。

Mesenchymal cells such as fibroblasts and myofibroblasts are the principle matrix-producing cells during pulmonary fibrogenesis. TGF β signaling plays an important role in stimulating the expression of type I collagen, the major component of extracellular matrix. Recently, a member of TGFβ superfamily, bone morphogenetic protein (BMP)-7, was reported to oppose TGFβ1 in fibrogenesis. Here, we have addressed the effects of gene transfer of BMP-7 on fibrogenic reaction using pulmonary myofibroblastic cell lines, in which collagen promoter activity was detected by luciferase assay. To do so, we first established cell lines from lung tissues of transgenic mice harboring the collagen 1A2 upstream sequence. These cell lines showed spindle shape, and expressed vimentin and aSMA, but not E-cadherin, which demonstrates that they are derived from lung myofibroblasts. They showed luciferase activity that was dose-dependent on TGFβ1 stimulation. This activity was also up-regulated by overexpression of Smad3 and down-regulated by that of Smad7. These cell lines were used to evaluate the effect of BMP-7 for following examination. In immunocytochemical examination, ectopic expression of BMP-7 (Ad-BMP-7) led to nuclear localization of phospho-Smadl/5/8 and suppressed that of Smad3. Ad-BMP-7 decreased collagen 1A2 promoter-dependent luciferase activity. Real-time RT PCR showed that Ad-BMP-7 suppressed the expression of collagen 1A2, tissue inhibitor of metalloproteinase and increased that of inhibitor of differentiation (Id) 2 and 3, which opposes TGFβ signaling. Furthermore, ectopic expression of Id2 and ID decreased collagen 1A2 promoter-dependent luciferase activity in pulmonary myofibroblasts. In conclusion, these data demonstrate that BMP-7 antagonizes TGFβ-dependent fibrogenesis by expression of 1d2 in mouse pulmonary myofibroblast cell lines.