的发展<em>体外</em>检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能
Summary
Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.
Abstract
Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor β, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor α. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.
Introduction
纤维化参与多种慢性进行性疾病，如间质性肺疾病，心脏纤维化，肝硬化，终端肾功能衰竭，系统性硬化，和自身免疫性疾病1的发病机制。间间质性肺疾病，特发性肺纤维化（IPF）是一种慢性进行性疾病，并显示预后差。 IPF的病理特点是成纤维细胞灶组成了与预后有关激活成纤维细胞和肌的发展。提出这样的肺成纤维细胞的起源是从几个间充质细胞，包括原驻地肺成纤维细胞和骨髓循环纤维细胞衍生的。最近，上皮-间质转化（EMT）已经提出了用间充质细胞2的形成相关联，并且有助于纤维化疾病的发病机制。
它被认为是电磁ŧ在胚胎发育过程中的重要作用，伤口愈合和癌症的进展，包括肿瘤侵袭和转移3。以下EMT的过程中，上皮细胞的上皮标记物，如E-钙粘蛋白的损失获得间充质细胞的能力，以及由间充质标志物，如波形蛋白和α平滑肌肌动蛋白（SMA）4,5的表达。以往的研究表明，EMT过程已与组织纤维化的发展在肾脏6和肺7被相关联的证据。此外，慢性炎症促进纤维化疾病8;此外，如炎性细胞因子如肿瘤坏死因子超家族成员14（TNFSF14; LIGHT），肿瘤坏死因子（TNF）-α和白细胞介素1β，已显示出提高的EMT 9-12。
胶原凝胶收缩试验，其中成纤维细胞嵌入型我一个基于胶原的细胞收缩测定胶原凝胶三维， 在体外模型收缩的评价理想。收缩是成纤维细胞的特征功能之一，并有助于正常的伤口修复和纤维化13。在该测定中，可以认为，成纤维细胞附着于I型通过整合素依赖性机制胶原被认为在一定条件下，以产生机械张力，并因此导致组织收缩。
这里，凝胶收缩测定的发展报告可以适于评估在经历EMT的细胞中采集收缩功能。这份报告表明，这种改进法是适用于评估在经历了EMT间质细胞收缩。
Protocol
Representative Results
Discussion
在这项研究中开发的协议包括两个步骤。进行第一步骤以诱导EMT的，而第二个步骤是在凝胶收缩试验。因为它确认细胞发生EMT是很重要的，第2步提供了一个极好的补充，形态学和基因表达的变化。以往的研究表明，A549细胞的EMT诱导仅24，TGF-β1;然而，正如我们以前10日报道，TNF-α处理增强EMT和收购的间充质细胞标志物。它被认为是TGF-β介导EMT的机制是SMAD依赖性25。 TNFα增?…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Tadashi Koyama for technical help. This work was supported in part by JSPS KAKENHI Grant Numbers 23249045, 15K09211, 15K19172; a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan; a grant for research on allergic disease and immunology, Japan.
Materials
| DMEM | sigma aldrich | 11965-092 | For A549 medium |
| FBS | GIBCO | 10437 | |
| Transforming Growth Factor-β1, Human, recombinant | Wako Laboratory chemicals | 209-16544 | |
| Recombinant Human TNF-α | R&D systems | 210-TA/CF | |
| E-Cadherin (24E10) Rabbit mAb | Cell Signaling Technology | #3195 | 1:3000 dilution |
| Vimentin (D21H3) Rabbit mAb | Cell Signaling Technology | #5741 | 1:3000 dilution |
| Anti-α-Tubulin antibody | sigma aldrich | T9026 | 1:10000 dilution |
| Monoclonal Anti-Actin, α-Smooth Muscle antibody | sigma aldrich | A5228 | 1:10000 dilution |
| Anti-N-cadherin antibody | BD Transduction Laboratories | #610920 | 1:1000 dilution |
| Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody) | GE Healthcare | NA931-100UL | 1:20000 dilution |
| Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody) | GE Healthcare | NA934-100UL | 1:20000 dilution |
| blocking reagent | GE Healthcare | RPN418 | 2% in TBS-T |
| 6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid | corning | #353046 | |
| 100 mm cell culture dish | TPP | #93100 | |
| DMEM, powder | life technologies | 12100-046 | For 4×DMEM |
| type 1 collagen gel | Nitta gelatin | Cellmatrix type I-A | |
| 24 well cell culture plate | AGC TECHNO GLASS | 1820-024 | |
| Gel Documentation System | ATTO | AE-6911FXN | Gel imager |
| gel analyzing software | ATTO | Densitograph, ver. 3.00 | analysing software bundled with AE-6911FXN |
| Trypsin-EDTA (0.05%), phenol red | life technologies | 25300054 | |
| 24 Well Plates, Non-Treated | IWAKI | 1820-024 | |
| Trypan Blue Solution, 0.4% | life technologies | 15250-061 | |
| RNA extraction kit | Qiagen | 74106 | |
| reverse transcriptase | life technologies | 18080044 | |
| real time PCR system | Stratagene | Mx-3000P | |
| SYBR green PCR kit | Qiagen | 204145 | |
| Protease Inhibitor Cocktail (100X) | life technologies | 78429 | |
| PVDF membrane | ATTO | 2392390 | |
| protein assay kit | bio-rad | 5000006JA | |
| polyacrylamide gel | ATTO | 2331810 | |
| western blotting detection reagent | GE Healthcare | RPN2232 | |
| cold CCD camera | ATTO | Ez-Capture MG/ST | |
| Trypsin inhibitor | sigma aldrich | T9003-100MG | |
| Polyoxyethylene (20)Sorbitan Monolaurate | Wako Laboratory chemicals | 163-11512 | |
| polyoxyethylene (9) octyiphenyl ether | Wako Laboratory chemicals | 141-08321 |
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