克罗恩病狭窄上方黏膜中的转化生长因子β信号传导与基质金属蛋白酶

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

作者信息

Di Sabatino A, Jackson C L, Pickard K M, Buckley M, Rovedatti L, Leakey N A B, Picariello L, Cazzola P, Monteleone G, Tonelli F, Corazza G R, MacDonald T T, Pender S L

机构信息

First Department of Medicine, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura delle Malattie Infiammatorie Croniche Intestinali, University of Pavia, Pavia, Italy.

出版信息

Gut. 2009 Jun;58(6):777-89. doi: 10.1136/gut.2008.149096. Epub 2009 Feb 6.

DOI:10.1136/gut.2008.149096

PMID:19201776

Abstract

BACKGROUND AND AIMS

In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD).

METHODS

Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration.

RESULTS

TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut.

CONCLUSIONS

Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

摘要

背景与目的

转化生长因子β（TGFβ）除了在抑制肠道组织损伤性免疫反应中起关键作用外，还是一种强效的促纤维化因子，可诱导胶原蛋白合成并调节基质降解基质金属蛋白酶（MMPs）及其抑制剂（TIMPs）之间的平衡。通过分析克罗恩病（CD）患者狭窄部位上方黏膜中的Smad蛋白和MMPs/TIMPs来研究TGFβ信号通路。

方法

从纤维狭窄型CD患者狭窄和非狭窄肠道上方的宏观正常黏膜中采集标本。将分离出的肌成纤维细胞与抗TGFβ阻断抗体或TGFβ1一起培养。通过定量逆转录聚合酶链反应（RT-PCR）分析TGFβ转录本。通过免疫印迹法测定Smad蛋白和MMPs。通过实时MMP-12活性测定法测量MMP-12活性。使用体外伤口愈合划痕试验评估肌成纤维细胞迁移。

结果

狭窄部位上方黏膜中的TGFβ转录本、磷酸化Smad2-Smad3（pSmad2-3）和TIMP-1蛋白高于非狭窄部位上方的黏膜。相比之下，狭窄肠道上方的黏膜中Smad7、MMP-12和MMP-3的表达较低。与非狭窄肠道上方黏膜的肌成纤维细胞相比，狭窄肠道上方黏膜的肌成纤维细胞显示出更高的TGFβ转录本、对TGFβ的pSmad2-3反应更强、TIMP-1增加、Smad7降低、胶原蛋白产生增加以及迁移能力降低。TGFβ阻断增加了肌成纤维细胞MMP-12的产生和迁移，与狭窄肠道相比，从非狭窄肠道上方黏膜分离的肌成纤维细胞中这种作用更明显。