開発<em>インビトロ</em>アッセイは、間葉細胞受けた上皮間葉移行の収縮機能を評価します
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
線維症は、間質性肺疾患、心臓線維症、肝硬変、末期腎不全、全身性硬化症、および自己免疫疾患の1のような種々の慢性進行性疾患の病因に関与しています。間質性肺炎のうち、特発性肺線維症（IPF）は慢性進行性疾患であり、予後不良を示しています。 IPFの病理学的特徴は予後と関連している活性化された線維芽細胞および筋線維芽細胞からなる線維芽細胞巣の開発です。このような肺の線維芽細胞の起源は、もともと常駐肺線維芽細胞及び骨髄から循環する線維細胞を含むいくつかの間葉系細胞に由来することが提案されています。最近、上皮間葉移行（EMT）は、間葉細胞2の形成に関連することが提案されている、および線維性疾患の病因に貢献します。
これは、と考えられているEMTは、腫瘍の浸潤および転移3を含む胎児の発育、創傷治癒、および癌の進行の過程で重要な役割を果たしています。 EMTのプロセスに続いて、上皮細胞は、E-カドヘリンなどの上皮マーカーの損失により、間葉系細胞の能力を取得し、そのようなビメンチンのような間葉マーカー、およびα平滑筋アクチン（SMA）4,5の発現による。以前の研究は、EMTプロセスは腎臓6および肺7における組織線維症の発症に関連しているという証拠を示しました。さらに、慢性炎症は、線維性疾患8を促進します 。さらに、腫瘍壊死因子スーパーファミリーメンバー14（TNFSF14、LIGHT）などの炎症性サイトカイン、腫瘍壊死因子（TNF）-α、インターロイキン1βは、EMT 9-12を増強することが示されています。
コラーゲンゲル収縮アッセイ線維芽細胞は、I型に埋め込まれたコラーゲンベースの細胞収縮アッセイコラーゲンゲル三次元、収縮性を評価するためのインビトロモデルにおける理想的です。収縮は、線維芽細胞の特徴的な機能の一つであり、正常な創傷修復および線維症13に貢献しています。このアッセイでは、線維芽細胞の付着は、いくつかの条件の下で機械的張力を生成することになっている私はインテグリン依存性のメカニズムを介して型コラーゲン、その結果、組織の収縮につながると考えられています。
ここで、ゲル収縮アッセイの開発は、EMTを受けた細胞における収縮機能の獲得を評価するように適合されることが報告されています。このレポートには、この修正されたアッセイは、EMTを経た間葉細胞で収縮性を評価するのに適していることを示しています。
Protocol
Representative Results
Discussion
本研究で開発されたプロトコルは、2つのステップを含みます。最初のステップは、第二段階は、ゲル収縮アッセイがあるが、EMTを誘導するために行われます。それは、細胞がEMTを受けたことを確認することが重要であるので、ステップ2は、形態学的および遺伝子発現の変化に優れた補数を提供します。以前の研究は、A549細胞のEMTは、TGF-β1のみ24によって誘導されたことを示しまし?…
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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