Desenvolvimento de um<em> In Vitro</em> Ensaio para avaliar contrátil Função de células mesenquimais que a transição epitelial-mesenquimal Sofreu
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
Fibrose está envolvida na patogénese de várias doenças progressivas, crónicas, tais como doença intersticial pulmonar, fibrose cardíaca, cirrose hepática, insuficiência renal terminal, esclerose sistémica, doença auto-imune e uma. Entre as doenças pulmonares intersticiais, fibrose pulmonar idiopática (PIF) é uma doença progressiva crónica e mostra mau prognóstico. marco patológico da FPI é o desenvolvimento de focos fibroblásticos consistindo de fibroblastos activados e miofibroblastos que estão associados com o prognóstico. As origens de tais fibroblastos pulmonares são propostos para ser derivadas de várias células mesenquimais, incluindo fibroblastos pulmonares originalmente residentes e fibrócitos que circulam a partir de medula óssea. Recentemente, tem sido proposto transição epitelial-mesenquimal (EMT) para ser associada com a formação de células mesenquimais 2, e para contribuir para a patogénese de doenças fibróticas.
Pensa-se que o EMT desempenha um papel importante no processo de desenvolvimento fetal, a cicatrização de feridas, e progressão do cancro, incluindo a invasão do tumor e metástases 3. Seguindo o processo de EMT, células epiteliais obter a capacidade das células mesenquimais por perda de marcadores epiteliais, tais como E-caderina, e através da expressão de marcadores de mesenquimais, tais como, a vimentina e α-actina de músculo liso (SMA) 4,5. Estudos anteriores demonstraram a evidência de que EMT processo tem sido associada com o desenvolvimento de tecido fibroso no rim e pulmão 6 7. Além disso, a inflamação crónica promove doença fibrótica 8; Além disso, tais citocinas inflamatórias como o factor de necrose tumoral membro da superfamília 14 (TNFSF14; LUZ), -α factor de necrose tumoral (TNF) e interleucina-1β, têm sido mostrados para melhorar EMT 9-12.
ensaio de contracção de gel de colagénio, um ensaio de contracção celular à base de colagénio, em que os fibroblastos são incorporados no tipo Igel de colagénio tridimensional, é um modelo in vitro ideal para a avaliação da contractilidade. Contractilidade é uma das funções características de fibroblastos e contribui para a reparação de feridas normal e fibrose 13. Neste ensaio, pensa-se que a fixação de fibroblastos ao colágeno tipo I através de mecanismos dependentes de integrina é suposto para produzir tensão mecânica sob certas condições, e, consequentemente, levar à contração do tecido.
Aqui, o desenvolvimento do ensaio de contracção de gel é referido como sendo adaptado para avaliar a aquisição da função contráctil nas células que foram submetidos a EMT. Este relatório demonstra que este ensaio modificado é adequado para avaliar a contratilidade em células mesenquimais que foram submetidos a EMT.
Protocol
Representative Results
Discussion
O protocolo desenvolvido neste estudo compreende duas etapas. O primeiro passo é realizado para induzir EMT, enquanto o segundo passo é o ensaio de contracção do gel. Uma vez que é importante para confirmar que as células foram submetidos a EMT, passo 2 proporciona um excelente complemento para as alterações morfológicas e expressão de genes. Estudos anteriores mostraram que as células de EMT A549 foi induzida por apenas 24 TGF-β1; No entanto, como já relatado anteriormente 10, o trata…
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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