Summary

Ontwikkeling van een<em> In Vitro</em> Assay om contractiele functie van mesenchymale Cellen die Onderging-mesenchymale transitie Evalueer

Published: June 10, 2016
doi:

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 is betrokken bij de pathogenese van verschillende chronische progressieve ziekten zoals interstitiële longziekte, cardiale fibrose, levercirrose, terminale nierinsufficiëntie, systemische sclerose en auto-immuunziekte 1. Onder interstitiële longziekten, idiopathische longfibrose (IPF) is een chronische progressieve ziekte en geeft een slechte prognose. Pathologische kenmerk van IPF is de ontwikkeling van fibroblastische foci bestaande uit geactiveerde fibroblasten en myofibroblasten die worden geassocieerd met de prognose. De oorsprong van deze pulmonaire fibroblasten voorgesteld kunnen worden uit verschillende mesenchymale cellen, zoals oorspronkelijk resident pulmonaire fibroblasten en circuleert fibrocytes uit beenmerg. Recentelijk heeft epitheliale-mesenchymale transitie (EMT) voorgesteld worden geassocieerd met de vorming van mesenchymale cellen 2, en bijdragen aan de pathogenese van fibrotische aandoeningen.

Er wordt gedacht dat EMT speelt een belangrijke rol in het proces van foetale ontwikkeling, wondgenezing en progressie van kanker, waaronder tumorinvasie en metastase 3. Na het proces van EMT, epitheelcellen verkrijgen het vermogen van mesenchymale cellen door verlies van epitheliale merkers, zoals E-cadherine, en expressie van mesenchymale merkers zoals vimentine en α-gladde spier actine (SMA) 4,5. Eerdere studies toonden het bewijs dat EMT proces is geassocieerd met de ontwikkeling van weefsel fibrose in de nier en de longen 6 7. Bovendien, chronische ontsteking bevordert fibrotische ziekte 8; Bovendien, zoals inflammatoire cytokinen zoals tumornecrosefactor superfamilieelement 14 (TNFSF14, LIGHT), tumornecrosefactor (TNF) -α, en interleukine-1β, is aangetoond EMT 9-12 verbeteren.

Collageengel contractie assay, een collageen gebaseerde celcontractie assay waarin fibroblasten zijn ingebed in type Icollageengel driedimensionaal, ideaal in vitro model voor de evaluatie van de contractiliteit. Contractiliteit is een van de karakteristieke functies van fibroblasten en draagt ​​bij normale wondgenezing en fibrose 13. Bij deze test wordt aangenomen dat de hechting van fibroblasten aan type I collageen door middel van integrine-afhankelijke mechanismen zou moeten mechanische spanning produceren onder bepaalde omstandigheden, en derhalve leiden tot weefselsamentrekking.

Hier wordt de ontwikkeling van de gel contractie assay gemeld worden aangepast aan de verwerving van contractiele functie uitwerkt in de cellen die EMT ondergingen. Dit rapport toont aan dat dit gewijzigde test is geschikt voor het evalueren van de contractiliteit in mesenchymale cellen die EMT ondergingen.

Protocol

1. Voorbereiding en Cultuur van longepitheelcellen Cultuur A549 humane long epitheelcellen (hechtende cellijn) in Dulbecco's Gemodificeerd Eagle Medium (DMEM) aangevuld met 10% foetaal runderserum (FBS), 100 IE / ml penicilline en 100 ug / ml streptomycine. Verwijder de celkweekmedia van kweekschaal en was eenmaal met 5-10 ml fosfaat gebufferde zoutoplossing (PBS). Na wassen onmiddellijk zuig het PBS. Voeg 2 ml trypsine / ethyleendiaminetetra-azijnzuur (EDTA) (0,05%) en incubeer bij 37…

Representative Results

Tijdens EMT, epitheelcellen verliezen epitheliale merkers, zoals E-cadherine, en krijgen de expressie van mesenchymale markers, zoals vimentine en α-gladde spier actine 4,5. Incubatie van A549 humane long epitheelcellen met TGF-β1 en TNF-α induceert EMT. Het uiterlijk van normale A549 cellen zijn geplaveide achtige vorm en driehoekig dat karakteristiek is voor epitheliale cellen (Figuur 3A), maar na gestimuleerd met TGF-β1 en TNF-α, het uiterlijk verander…

Discussion

De in deze onderzoeksprotocol omvat twee stappen. De eerste stap wordt uitgevoerd om EMT induceren, terwijl de tweede stap is de gel samentrekking assay. Aangezien het belangrijk te bevestigen dat cellen ondergingen EMT, stap 2 een uitstekende aanvulling op de morfologische veranderingen en gen expressie. Eerdere studies toonden aan dat EMT van A549-cellen werd geïnduceerd met slechts 24 TGF-β1; echter, zoals we eerder hebben gemeld 10, TNF-α behandeling verbetert EMT en de overname van mesenchy…

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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Cite This Article
Mikami, Y., Matsuzaki, H., Takeshima, H., Makita, K., Yamauchi, Y., Nagase, T. Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition. J. Vis. Exp. (112), e53974, doi:10.3791/53974 (2016).

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