Desarrollo de una<em> In Vitro</em> Ensayo para evaluar la función contráctil de las células mesenquimales que la transición epitelio-mesenquimal Sufrió
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
Fibrosis está implicada en la patogénesis de diversas enfermedades progresivas crónicas, como la enfermedad pulmonar intersticial, fibrosis cardiaca, cirrosis hepática, insuficiencia renal terminal, la esclerosis sistémica, y las enfermedades autoinmunes 1. Entre las enfermedades pulmonares intersticiales, fibrosis pulmonar idiopática (FPI) es una enfermedad progresiva crónica y muestra mal pronóstico. característica patológica de la IPF es el desarrollo de focos de fibroblasto que consiste en fibroblastos y miofibroblastos activados que están asociados con el pronóstico. Se proponen los orígenes de tales fibroblastos pulmonares que se derivan de varias células mesenquimales, incluyendo fibroblastos pulmonares residentes originalmente y que circulan fibrocitos de la médula ósea. Recientemente, se ha propuesto transición epitelio-mesenquimal (EMT) que se asocia con la formación de células mesenquimales 2, y para contribuir a la patogénesis de trastornos fibróticos.
Se cree que la EMT juega un papel importante en el proceso de desarrollo fetal, la cicatrización de heridas, y la progresión del cáncer, incluyendo la invasión tumoral y la metástasis 3. Tras el proceso de EMT, las células epiteliales obtener la capacidad de las células mesenquimales por la pérdida de marcadores epiteliales, tales como la E-cadherina, y por la expresión de marcadores mesenquimales, tales como la vimentina, y α-actina de músculo liso (SMA) 4,5. Estudios anteriores mostraron la evidencia de que proceso de EMT se ha asociado con el desarrollo de fibrosis de tejidos en el riñón y el pulmón 6 7. Además, la inflamación crónica promueve la enfermedad fibrótica 8; Además, las citoquinas inflamatorias tales como miembro de la superfamilia de factor de necrosis tumoral 14 (TNFSF14; LIGHT), factor de necrosis tumoral (TNF) -α, y la interleucina-1β, se ha demostrado para mejorar EMT 9-12.
ensayo de contracción de gel de colágeno, un ensayo de contracción de las células a base de colágeno en el que los fibroblastos se incrustan en el tipo Igel de colágeno tridimensional, es un ideal modelo in vitro para la evaluación de la contractilidad. La contractilidad es una de las funciones características de los fibroblastos y contribuye a la reparación de heridas normal y fibrosis 13. En este ensayo, se cree que la unión de los fibroblastos para colágeno tipo I a través de mecanismos dependientes de integrina se supone para producir tensión mecánica bajo ciertas condiciones, y en consecuencia dar lugar a la contracción del tejido.
Aquí, se informa que el desarrollo del ensayo de contracción de gel que adaptarse para evaluar la adquisición de la función contráctil en las células que se sometieron a EMT. Este informe demuestra que este ensayo modificado es adecuado para la evaluación de la contractilidad de las células mesenquimales que se sometieron a EMT.
Protocol
Representative Results
Discussion
El protocolo desarrollado en este estudio comprende dos etapas. La primera etapa se realiza para inducir EMT, mientras que el segundo paso es el ensayo de contracción de gel. Dado que es importante confirmar que las células se sometieron a EMT, el paso 2 proporciona un excelente complemento a los cambios en la expresión de genes y morfológicos. Estudios anteriores demostraron que la EMT de las células A549 fue inducida por sólo el 24 por TGF-β1; Sin embargo, como hemos informado anteriormente 10,<…
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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