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

腺病毒载体选择性表达基因调控治疗肺纤维化的基础研究

• 批准号: 15591485
• 负责人: INOUE Kiyotoshi
• 金额: $ 2.24万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591485/
• 关键词: pulmonary fibrosis; Cre; loxP system; adenovirus vector; fibroblast; alveolar epithelial cell; BMP7; Id2; Id3; ld2; ld3; TK; Akt; YB-1

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.

成纤维细胞和肌成纤维细胞等间充质细胞是肺纤维化形成过程中产生基质的主要细胞。转化生长因子β信号在刺激细胞外基质主要成分I型胶原的表达中起重要作用。最近研究发现，转化生长因子β超家族成员骨形态发生蛋白-7可拮抗转化生长因子β-1在纤维化形成中的作用。在这里，我们研究了BMP-7基因转移对肺成纤维细胞系纤维化反应的影响，其中胶原启动子的活性通过荧光素酶活性检测。为此，我们首先从含有胶原1A2上游序列的转基因小鼠的肺组织中建立了细胞系。这些细胞系呈纺锤形，表达波形蛋白和ASMA，但不表达E-钙粘蛋白，表明它们来源于肺成纤维细胞。它们的荧光素酶活性与转化生长因子β-1刺激呈剂量依赖关系。这种活性也被Smad3的过表达上调，而被Smad7的过表达下调。这些细胞系被用来评价BMP-7的作用，为后续的检测做准备。在免疫细胞化学检测中，异位表达BMP-7(Ad-BMP-7)导致磷酸化Smad1/5/8的核定位，抑制Smad3的核定位。Ad-BMP-7可降低胶原1A2启动子依赖的荧光素酶活性。实时逆转录聚合酶链式反应显示，Ad-BMP-7可抑制基质金属蛋白酶组织抑制因子胶原1A2的表达，增加与转化生长因子β信号转导相反的分化抑制因子2和3的表达。此外，Id2和Id2的异位表达降低了肺成纤维细胞中胶原1A2启动子依赖的荧光素酶活性。综上所述，这些结果表明骨形态发生蛋白-7通过在小鼠肺成纤维细胞系中表达1d2来拮抗转化生长因子β依赖的纤维化形成。

项目成果

THE EPIDEMIOLOGY OF INTERSTITIAL LUNG-DISEASES

• DOI: 10.1164/ajrccm.150.4.7921471
• 发表时间: 1994-10-01
• 期刊: AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
• 影响因子: 24.7
• 作者: COULTAS, DB;ZUMWALT, RE;SOBONYA, RE
• 通讯作者: SOBONYA, RE

Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts.

缺氧调节转化生长因子-β亚型对原代人肺成纤维细胞基质形成的影响。

• 发表时间: 2003
• 期刊: Cytokine 24
• 作者: Papakonstantinou E;Aletras AJ;Roth M;Tamm M;Karakiulakis G.
• 通讯作者: Karakiulakis G.

Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3.

原代人肺成纤维细胞响应 TGF-β1 和 TGF-β3 的细胞外基质沉积。

• 发表时间: 1999
• 期刊: Am J Physiol 276
• 作者: Eickelberg O;Kohler E;Reichenberger F;Bertschin S;Woodtli T;Erne P;Perruchoud AP;Roth M.
• 通讯作者: Roth M.

Smad3 null mice develop airspace enlargement and are resistant to TGF-beta-mediated pulmonary fibrosis.

Smad3缺失小鼠的空腔扩大并且对TGF-β介导的肺纤维化具有抵抗力。

• 发表时间: 2004
• 期刊: J Immunol 173
• 作者: Bonniaud P;Kolb M;Galt T;Robertson J;Robbins C;Stampfli M;Lavery C;Margetts PJ;Roberts AB;Gauldie J.
• 通讯作者: Gauldie J.

Two-step gene transfer using an adenoviral vector carrying the CD4 gene and human immunodeficiency viral vectors.

使用携带 CD4 基因的腺病毒载体和人类免疫缺陷病毒载体进行两步基因转移。

• 发表时间: 1996
• 期刊: Hum Gene Ther 77
• 作者: Miyake K;Tohyama T;Shimada T.
• 通讯作者: Shimada T.