Utveckling av en<em> In Vitro</em> Analys för att utvärdera kontraktila funktion av mesenkymala celler som genomgick Epithelial-Mesenkymala Övergångs
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
Fibros är involverat i patogenesen för olika kroniska progressiva sjukdomar, såsom interstitiell lungsjukdom, hjärtfibros, levercirros, terminal njursvikt, systemisk skleros och autoimmun sjukdom 1. Bland interstitiell lungsjukdom, är idiopatisk lungfibros (IPF) en kronisk progressiv sjukdom och visar dålig prognos. Patologiskt kännemärke för IPF är utvecklingen av fibroblastisk foci som består av aktiverade fibroblaster och myofibroblaster som är associerade med prognosen. föreslagna Ursprunget till sådana lung fibroblaster komma från flera mesenkymala celler, inklusive ursprungligen bosatta lung fibroblaster och cirkulerande fibrocyter från benmärg. Nyligen har epitelial-mesenkymala övergång (EMT) föreslagits för att vara associerade med bildningen av mesenkymala celler 2, och att bidra till patogenesen av fibrotiska störningar.
Man tror att EMT spelar viktiga roller i processen för fosterutveckling, sårläkning och utvecklingen av cancer, inklusive tumörinvasion och metastas 3. Följande förfarandet av EMT, epitelceller erhålla förmågan hos mesenkymala celler genom förlust av epitel-markörer, såsom E-cadherin, och genom uttryck av mesenkymala markörer, såsom vimentin, och α-glattmuskelaktin (SMA) 4,5. Tidigare studier visade tecken på att EMT process har associerats med utveckling av vävnadsfibros i njuren 6 och lunga 7. Dessutom främjar kronisk inflammation fibrotisk sjukdom 8; dessutom sådana inflammatoriska cytokiner såsom Tumörnekrosfaktor superfamiljmedlem 14 (TNFSF14; LIGHT), tumörnekrosfaktor (TNF) -α, och interleukin-1β, varit har visat sig förstärka EMT 9-12.
Kollagengel kontraktion analys en kollagenbaserad cell kontraktion analys i vilken fibroblaster är inbäddade i typ Ikollagengel tredimensionellt, är en idealisk in vitro-modell för utvärdering av kontraktilitet. Kontraktilitet är ett av de karakteristiska funktionerna hos fibroblaster och bidrar till normal sårläkning och fibros 13. I denna analys är det tänkt att fastsättningen av fibroblaster till typ I-kollagen genom integrin-beroende mekanismer är tänkt för att producera mekanisk spänning under vissa betingelser, och följaktligen leda till vävnadssammandragning.
Här, är utvecklingen av gelén kontraktion assay rapporterats vara anpassad för att utvärdera förvärv av kontraktil funktion i de celler som genomgick EMT. Denna rapport visar att denna modifierade analys är lämplig för att utvärdera kontraktiliteten i mesenkymala celler som genomgick EMT.
Protocol
Representative Results
Discussion
Det protokoll som utvecklats i denna studie består av två steg. Det första steget utförs för att inducera EMT, medan det andra steget är den gelkontraktion analysen. Eftersom det är viktigt att bekräfta att celler genomgick EMT, steg 2 ger ett utmärkt komplement till de morfologiska och genuttryck förändringar. Tidigare studier har visat att EMT av A549-celler inducerades med endast 24 TGF-β1; Men, som vi tidigare 10 har rapporterats förstärker TNF-α behandling EMT och förvärvet av…
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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