콜론-26 암 종양 베어링 암 악액질의 연구를위한 모델로 마우스를
Summary
Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.
Abstract
Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.
Introduction
근육 낭비는 암, 패혈증, 간, 간경변, 심장 및 신장 질환, 만성 폐쇄성 폐 질환, 에이즈 등의 다양한 임상 조건의 심각한 합병증이다. 특히, 근육 쇠약 한 암 환자의 50 % 이상에서 명백하다. 인해 및 / 또는로부터 골격근 단백질 분해 시스템의 과도한 활성화로 증가 단백질 분해로부터 암 결과 골격근 상실 단백질 합성 (6) 감소 하였다. 지방 분해는 지방 조직의 붕괴를 초래 또한 명백하다. 임상 악액질 감소 된 품질 및 수명의 길이와 연관된 20 사망 원인으로 추정된다 – 암 환자 (7)의 30 %. 최대한 가깝게 인간 질환과 유사한 실험 모델의 사용은 유익 할 것이다. 최적의 동물 모델 높은 재현성과 다를 치료에서 제한된 간섭 및 예측 인자 특징일반적으로 임상 상태 (8)과 관련된 다이어트, 성별, 유전 적 배경. 새로운 방법은 암의 발달에 민감한 유전자 변형 마우스를 사용하는 것이지만, 지금까지 암 악액질은 암 세포 또는 발암 주입 이식 특징 동물 모델에서 주로 연구되어왔다.
C26 암종 베어링 마우스 암 악액질 2,5의 잘 특징으로 널리 사용 모델을 나타내는 (도 26 – 결장 선암 함). 주로 고급 지방 및 단백질 이화 9 체 근육 감량 C26에서 종양의 성장 결과. 일반적으로, 몸 전체 중량 대비 10 % 종양 중량은 골격근 질량 20-25 %의 감소 및 지방 3,10의 큰 고갈과 연관된다. 비장 비대 및 또한 급성기 반응의 활성화 및 프로 infla의 높이와 함께, 종양 성장을 관찰mmatory 사이토 카인 수준 3,11. 이들 중, 이곳은 IL-6 사이토 카인이 아마 악액질 (12)의 유일한 유도제없는 경우에도 C26 모델에서 근육 쇠약을 매개에서 중요한 역할을하는 것으로 알려져있다. 상승 된 IL-6는 JAK / STAT3 경로의 활성화를 통해 근육 위축이 발생하고,이 전사 인자를 억제하는 근육 3,4- 낭비를 방지 할 수있다.
C26에 의한 근육 낭비하는 동안, 근육 위축의 많은 조건에서와 같이 근육 질량이없는 세포 사멸 또는 섬유 (13)의 손실을 크게 근육 섬유에서 근육 단백질 함량의 감소를 통해 손실됩니다. C26 악액질에서 작은 단면적을 향한 이동은 당분 및 산화 섬유 둘 모두에서 관찰된다. 이는 또한 감소 된 근력 5와 일치한다. 전세계 여러 그룹은 암 CAC 대한 근 소모성 새로운 중재자 또는 임상 적 약물을 발견하기 위해 C26 모델의 장점을 찍은hexia. 그러나,이 모델의 사용을위한 다양한 방법이 획득 된 데이터의 일관성에 관한 문제를 제기하고 다른 실험 조건에서 재현성 장벽 포즈보고되었다. 여기에서 우리는 표준화 및 재현성 데이터를 산출 암 악액질의 연구를위한이 모델의 전형적인 사용을보고합니다.
Protocol
Representative Results
Discussion
특히 최근의 단계에서, 결장암이 불량한 결과 및 환자의 삶의 질에서의 감소에 대한 책임이 악액질의 발전과 관련된다. 많은 연구는 암에 이차적 상태의 치료에 초점을 맞추고있다; 그러나,이 방향으로 많은 노력에도 불구하고, 여전히 암 악액질 (21)에 대한 승인 된 치료가 없습니다. 동물 모델 결과의 번역을 최대화하기 위해 가능한 한 가깝게 인간 병리 닮은 따라서, 필수적이다.
<p cl…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Richard Lieber and Shannon Bremner for their ImageJ macro and instructions. While at Thomas Jefferson University, this work was supported by the Pennsylvania Department of Health CURE Grant TJU No. 080-37038-AI0801. Subsequently, this study was supported by a grant to AB from the National Institutes of Health (R21CA190028), and by grants to TAZ from the National Institutes of Health (R01CA122596, R01CA194593), the IU Simon Cancer Center, the Lustgarten Foundation, the Lilly Foundation, Inc., and the IUPUI Pancreas Signature Center.
Materials
| Cell culture Flasks | Falcon – Becton Dickinson | 35-5001 | |
| DMEM | Cellgro | 10-017-CV | |
| FBS | Gibco | 26140 | |
| Streptomycin-Penicillin | Cellgro | 30-002-CI | |
| CD2F1 mice | Harlan | 060 | |
| Anesthesia apparatus | EZ-Anesthesia | EZ-7000 | |
| 2-Methyl Butane | Sigma-Aldrich | M32631 | |
| OCT | Tissue-Tek | 4583 | |
| Cryostat | Leica | CM1850 | |
| Cork disks | Electron Microscopy Sciences | 63305 | |
| Superfrost plus glass slides | VWR | 48311-703 | |
| Anti-Laminin Rabbit polyclonal antibody | Sigma-Aldrich | L9393 | |
| Anti-Dystrophin Mouse Monoclonal antibody | Vector Laboratories | VP-D508 | |
| Alexa Flour 594 anti-mouse IgG | Life Technologies | A11062 | |
| Alexa Flour 594 anti-rabbit IgG | Life Technologies | A21211 | |
| Hematoxylin | Sigma-Aldrich | GHS216 | |
| Eosin | Sigma-Aldrich | HT110332 | |
| Xylene | Acros Organics | 422680025 | |
| Cytoseal-XYL | Thermo | 8312-4 | |
| Microscope | Zeiss | Observer.Z1 | |
| Bamboo Tablet | Wacom | CTH-661 | |
| Prism 7.0 for Mac OS X | GraphPad Software, Inc. | ||
| Excel for Mac 2011 | Microsoft Corp. | ||
| Image J | US National Institutes of Health | IJ1.46 | http://rsbweb.nih.gov/ij/download.html |
| Microtainer | BD | 365873 |
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