Bir Gelişimi<em> İn Vitro</em> Deney Mezenkimal Hücreleri Uyguladığımız Epitel-Mezenkimal Geçiş kasılma Fonksiyonu değerlendirin
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
Fibroz, interstisyel akciğer hastalığı, kardiyak fibroz, karaciğer sirozu, böbrek yetmezliği, terminal, sistemik skleroz, otoimmün hastalık 1 gibi çeşitli kronik progresif hastalıkların patogenezinde rol oynamaktadır. interstisyel akciğer hastalıkları arasında, idiyopatik pulmoner fibrozis (IPF) kronik ilerleyici bir hastalıktır ve kötü prognozu gösterir. IPF patolojik bulgusudur prognoz ile ilişkili olduğu aktive fibroblast ve miyofibroblastlar oluşan fibroblastik odaklar geliştirilmesidir. Bu tür akciğer fibroblast kökenleri aslen ikamet akciğer fibroblastlar dahil olmak üzere ve kemik iliği fibrositlerden dolaşan birkaç mezenkimal hücrelerden, elde edilecek önerilmektedir. Son zamanlarda, epitelyal mezenkimal geçişi (EMT) mezenkimal hücreler 2 oluşumu ile ilişkili olduğu öne sürülmüştür ve fibrotik hastalıkların patogenezine katkıda bulunmak.
Bu düşünülmektedir EMT tümör invazyonu ve metastaz 3 olmak üzere fetal gelişim sürecinde önemli roller, yara iyileşmesi ve kanserin ilerlemesini, oynar. EMT süreci takiben, epitel hücreler, E-kadherin gibi epitelyal belirteçleri kaybıyla mezenkimal hücrelerin yeteneği elde etmek ve örneğin vimentin gibi mezenşimal belirteçleri ve α-düz kas aktini (SMA) 4,5 ekspresyonuyla. Önceki çalışmalar EMT süreç böbrek 6 ve akciğer 7 doku fibrozis gelişimi ile ilişkili olduğunu kanıt gösterdi. Ayrıca, kronik inflamasyon fibrotik hastalığı 8 teşvik; Bundan başka, tümör nekroz faktörü üst aile üyesini 14 (TNFSF14; LIGHT) gibi enflamatuar sitokinler, tümör nekroz faktörü (TNF) -a ve interlökin-1, EMT 9-12 geliştirmek için gösterilmiştir.
Kollajen jel daralma tahlili, fibroblastlar tip I gömülü olduğu bir kollajen bazlı hücre kasılması deneyikolajen jeli üç boyutlu, kontraktilite değerlendirilmesi için in vitro model bir idealdir. Kontraktilite fibroblast karakteristik işlevlerinden biridir ve normal yara onarımı ve fibrozis 13 katkıda bulunur. Bu deneyde, fibroblast ek bazı koşullar altında, mekanik gerilim üretilmesi için gereken I integrin bağlantılı mekanizmalar vasıtasıyla kolajen tip, ve bunun sonucu olarak doku daralmaya yol düşünülmektedir.
Burada, jel daralma deneyinin geliştirilmesi EMT yapılan hücrelerde kontraktil fonksiyonun satın değerlendirmek üzere uyarlanmış olduğu bildirilmektedir. Bu raporda, bu modifiye edilmiş deney EMT uygulanan mezenkimal hücreler kontraktiliteııin değerlendirilmesi için uygun olduğunu göstermektedir.
Protocol
Representative Results
Discussion
Bu çalışmada geliştirilen protokol iki adımları içerir. İlk adım, ikinci bir basamaklı bir jel tahlili daralma ise, EMT indüklemek için gerçekleştirilir. hücrelerin EMT uygulanan onaylamak için önemli olduğundan, adım 2 morfolojik ve gen ekspresyon değişiklikleri için mükemmel bir tamamlayıcı sağlar. Daha önceki çalışmalar, A549 hücrelerinin EMT, TGF-ß1 sadece 24 tarafından indüklendiğini göstermiştir; Daha önce 10 rapor ettiği gibi, ancak, TNF-α tedavisi EMT…
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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