Développement d'un<em> In vitro</em> Essai pour évaluer contractile Fonction mésenchymateuses Les cellules de transition épithéliale-mésenchymateuse A subi
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
La fibrose est impliquée dans la pathogenèse de diverses maladies chroniques évolutives telles que la maladie pulmonaire interstitielle, la fibrose cardiaque, la cirrhose du foie, une insuffisance rénale terminale, la sclérose systémique, la maladie auto – immune et 1. Parmi les maladies pulmonaires interstitielles, fibrose pulmonaire idiopathique (IPF) est une maladie chronique progressive et montre un mauvais pronostic. caractéristique pathologique du IPF est le développement de foyers fibroblastique constitué par les fibroblastes et les myofibroblastes activés qui sont associées au pronostic. Les origines de ces fibroblastes pulmonaires sont proposés pour être dérivé de plusieurs cellules mésenchymateuses, y compris les fibroblastes pulmonaires résidents à l'origine et en circulation fibrocytes depuis la moelle osseuse. Récemment, la transition épithéliale-mésenchymateuse (TEM) a été proposé d'associer à la formation de cellules mésenchymateuses 2, et de contribuer à la pathogenèse des troubles fibrotiques.
On pense que la surveillance électroniqueT joue un rôle important dans le processus de développement du foetus, la cicatrisation des plaies et la progression du cancer, y compris l' invasion tumorale et les métastases 3. En suivant le procédé de l' EMT, les cellules épithéliales obtenir la capacité des cellules mésenchymateuses par la perte de marqueurs épithéliaux, tels que la E-cadhérine, et par l' expression de marqueurs de mésenchymateuses, comme la vimentine, et α-actine du muscle lisse (SMA) 4,5. Des études antérieures ont montré des preuves de ce processus EMT a été associé au développement de la fibrose tissulaire au niveau du rein et du poumon 6 7. En outre, l' inflammation chronique favorise les maladies fibrotiques 8; En outre, de telles cytokines pro – inflammatoires comme le facteur de nécrose tumorale membre de la superfamille 14 (TNFSF14, la lumière), le facteur de nécrose tumorale (TNF) -α, l' interleukine-1β et, il a été démontré pour améliorer EMT 12/09.
test de la contraction du gel de collagène, une contraction de la cellule test à base de collagène dans lequel les fibroblastes sont noyés dans le type Igel de collagène dans les trois dimensions, est un idéal modèle in vitro pour l'évaluation de la contractilité. Contractilité est l' une des fonctions caractéristiques des fibroblastes et contribue à la réparation de plaies et de la fibrose 13 normale. Dans cet essai, on pense que la fixation des fibroblastes du collagène de type I par des mécanismes de intégrine-dépendant est censé produire une tension mécanique dans certaines conditions, et par conséquent, conduire à une contraction des tissus.
Ici, la mise au point du test de contraction du gel est signalé à être adapté pour évaluer l'acquisition de la fonction contractile dans les cellules qui ont subi EMT. Ce rapport démontre que cet essai modifié est adapté pour évaluer la contractilité dans les cellules mésenchymateuses qui ont subi EMT.
Protocol
Representative Results
Discussion
Le protocole mis au point dans la présente étude comprend deux étapes. La première étape est réalisée pour induire EMT, tandis que la deuxième étape est l'essai de contraction du gel. Comme il est important de confirmer que les cellules ont subi EMT, l'étape 2 fournit un excellent complément aux changements d'expression morphologiques et génétiques. Des études antérieures ont montré que des cellules EMT A549 a été induite par le TGF-β1 seulement 24; Cependant, comme nous l' …
Disclosures
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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