개발<em> 체외</em> 분석은 중간 엽 세포를 시행 한 상피 - 중간 엽 전환의 수축 기능을 평가하는
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
섬유화 간질 성 폐 질환, 심장 섬유증, 간경변, 단말 신부전, 전신성 경화증,자가 면역 질환 한 각종 만성 진행성 질환의 발병에 관여된다. 간질 성 폐 질환 중 특발성 폐 섬유증 (IPF)은 만성 진행성 질환이며, 불량한 예후를 보여줍니다. IPF의 병리학 적 특징은 예후와 관련된 활성화 된 섬유 아세포 및 근섬유로 이루어진 섬유 아세포 초점의 개발이다. 이러한 폐 섬유 아 세포의 기원은 원래 주민 폐 섬유 아 세포를 포함하고 골수에서 섬유 세포를 순환 여러 중간 엽 세포에서 파생 할 것을 제안한다. 최근 상피 간엽 전이 (EMT)는 간엽 세포 (2)의 형성과 관련이 제안되었으며, 섬유 성 질환의 발병에 기여.
그것은 생각된다 EMT는 종양의 침윤과 전이 3를 포함하여 태아의 발달 과정에서 중요한 역할을, 상처 치유, 암의 진행을한다. EMT의 방법에 따라, 상피 세포는 E-cadherin의 상피 마커의 손실에 의해 중간 엽 세포의 능력을 획득하고, 그러한 멘틴 같은 중배엽 마커, 및 α-평활근 액틴 (SMA) -4,5- 의해 발현. 이전 연구 EMT 처리가 신장 6 및 7에서 폐 섬유증 조직의 발달과 관련되었다는 증거를 나타내었다. 또한, 만성 염증은 섬유 성 질환 (8)을 촉진; 또한, 종양 괴사 인자 상과 부재 (14) (TNFSF14; LIGHT)와 같은 염증성 사이토 카인, 종양 괴사 인자 (TNF) -α 및 인터루킨 1β는 EMT 9-12을 향상시키는 것으로 나타났다.
콜라겐 겔 수축 분석법, 섬유 아세포 타입 I에 포함 된 콜라겐 계 세포 수축 분석법콜라겐 겔 삼차원, 수축의 평가를위한 시험 관내 모델에서 이상적이다. 수축성 섬유 아세포의 특성 함수 중 하나이며 정상 상처 복구 및 섬유증 (13)에 기여한다. 이 분석에서, 섬유 아세포의 부착이 어떤 조건 하에서 기계적 장력을 생성하도록되어 I는 인테그린 의존성 메커니즘을 통해 입력 콜라겐, 결과적으로 조직의 수축을 유도하는 것으로 생각된다.
여기서, 겔 수축 분석법의 개발이 EMT을받은 세포의 수축 기능의 획득을 평가하도록 적응 될 것으로보고있다. 이 보고서는이 변형 분석이 EMT을 시행 한 중간 엽 세포의 수축력을 평가하기에 적합 함을 보여줍니다.
Protocol
Representative Results
Discussion
본 연구에서 개발 된이 프로토콜은 두 단계를 포함한다. 첫 번째 단계에서는 두 번째 단계는 겔 수축 분석법 동안, EMT를 유도하기 위해 실행된다. 이 세포는 EMT을받은 것을 확인하는 것이 중요하기 때문에, 2 단계는 형태 학적 및 유전자 발현의 변화에 훌륭한 보완을 제공합니다. 이전 연구 A549 세포 EMT는 TGF-β1을 24 만에 의한 것으로 나타났다; 우리는 이전에 10보고 그러나, TNF-…
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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