异型三维<em在体外</em>卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型
Summary
我们描述的方法建立<em在体外</em>异型三维模型，包括卵巢成纤维细胞和正常卵巢表面或卵巢癌上皮细胞。我们讨论了使用这些模型来研究间质 – 上皮细胞相互作用过程中发生卵巢癌的发展。
Abstract
卵巢上皮细胞癌（EOCS）是在西方社会的妇科恶性肿瘤死亡的首要原因。尽管手术治疗和改善铂类为基础的化疗方案，很少有改善在EOC存活率，超过四十年的1,2。虽然第一期肿瘤5年生存率> 85％，Ⅲ/Ⅳ期疾病的存活率为40％。因此，平机会的高发生率，死亡率可显着减少，如果检测到肿瘤在早期阶段，更可治疗，3-5。目前，疾病早期发展的分子遗传学和生物学基础，更是知之甚少。更具体地说，很少有人了解的微环境在肿瘤发生中的作用，但已知的危险因素的EOCS（ 如年龄和奇偶校验）表明，微环境中起着关键的作用的早期起源EOCS的。因此，我们开发了三维异型模型既正常卵巢和早期卵巢癌。而在正常卵巢中，我们共培养正常卵巢表面上皮细胞（IOSE）和正常间质成纤维细胞（INOF）细胞，永生化的retrovrial转导的催化亚基端粒酶全酶（hTERT）的延长寿命这些细胞在培养。为了模拟卵巢上皮细胞转化的CMYC IOSE细胞癌基因的过度表达，再次合作，培养与INOF细胞的最早阶段。这些异型上皮细胞的转化和侵袭的模型被用来研究老化和衰老的影响。在这里，我们描述了在发展的这些三维模型的方法步骤，这些方法是不正常卵巢和卵巢癌组织的发展，可用于研究其他类型的组织基质和上皮细胞间的相互作用是一个根本方面的组织维护和二sease发展。
Protocol
图1示出了概述下面描述的工作流程。 1。正常卵巢成纤维细胞在体外寿命的延长，过表达的端粒酶全酶的催化亚基分离卵巢组织可以收集患者有知情同意和批准的机构审查委员会（美国机构）。腹式全子宫切除术或腹腔镜子宫切除术，双侧输卵管卵巢切除术的正常卵巢组织可以收集。在这项研究中，组织由病理学家和卵巢基质活检的细胞培?…
Discussion
早期的上皮性卵巢癌（EOC）的生物学知之甚少。也许多年在这一领域的主要障碍之一是缺乏组织特异性疾病的起源，EOC发展的微环境中的作用的重要性的认识。在过去的几年中，已经很清楚，EOC是一种异质性疾病与多个的不同histophathological亚型，可能有不同的细胞起源不同亚型。例如，当前的数据表明，高品位的浆液EOCS可能源于双侧输卵管分泌性上皮细胞和卵巢表面上皮细胞，至少一定比例被认…
Declarações
The authors have nothing to disclose.
Acknowledgements
进行这项研究的凯克医学院，美国加州大学和伦敦大学学院，英国。 KL是由国家卫生研究所获得5 U19 CA148112-02。 BG是由前夕上诉妇科肿瘤慈善机构（英国）项目的资助。 / UCL在UCLH开展这项工作的一些部分部的健康的的NIHR生物医学研究中心资助计划的资助。
Materials
| Reagent | Supplier | Catalogue Number |
| PolyHEMA, suitable for cell culture | Sigma Aldrich | P3932 |
| Molecular biology grade ethanol | Sigma Aldrich | E7023 |
| Sterile water for cell culture | VWR | 12001-356 |
| MCDB105 | Sigma Aldrich | M6395 |
| Medium 199 | Sigma Aldrich | M2154 |
| Hyclone Fetal bovine serum | Thermo Scientific | SH30088.03 |
| Gentamicin | Sigma Aldrich | G1397 |
| Amphotericin B | Sigma Aldrich | A2942 |
| pBABE-hygro-hTERT | Addgene | 1773 |
| PBS | VWR | 12001-766 |
| 0.25% trypsin-EDTA | Invitrogen | 25200-072 |
| Cell strainer (40 or 70 μm) | VWR | 21008-949 21008-952 |
| Anti-fibroblast surface protein antibody, clone 1B10 | Sigma | F4771 |
| Anti-pan-cytokeratin antibody (C11) | Santa Cruz | sc-8018 |
| Polybrene (hexadimethrine bromide) | Sigma | 107689 |
| TeloTAGGG Telomerase PCR ELISAPLUS | Roche | 12013789001 |
| TeloTAGGG Telomere Length Assay | Roche | 12209136001 |
Table 1. Reagents and Equipment Referred to in this study.
Referências
- Jelovac, D., Armstrong, D. K. Recent progress in the diagnosis and treatment of ovarian cancer. CA Cancer J. Clin. , (2011).
- Office for National Statistics. . Cancer Statistics registrations: registrations of cancer diagnosed in 2008. , (2011).
- Köbel, M., Kalloger, S. E., Santos, J. L. Tumor type and substage predict survival in stage I and II ovarian carcinoma: insights and implications. Gynecol. Oncol. 116, 50-56 (2010).
- Köbel, M., Kalloger, S. E., Boyd, N. Ovarian carcinoma subtypes are different diseases: implications for biomarker studies. PLoS Med. 5, e232 (2008).
- Smith, L. H., Morris, C. R., Yasmeen, S. Ovarian cancer: can we make the clinical diagnosis earlier. Cancer. 104, 1398-1407 (2005).
- Aviv, A., Hunt, S. C., Lin, J., Cao, X., Kimura, M., Blackburn, E. Impartial comparative analysis of measurement of leukocyte telomere length/DNA content by Southern blots and qPCR. Nucleic Acids Res. 39, e134 (2011).
- Kim, N. W., Wu, F. Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP. Nucleic Acids Res. 25, 2595-2597 (1997).
- Lawrenson, K., Grun, B., Benjamin, E. Senescent fibroblasts promote neoplastic transformation of partially transformed ovarian epithelial cells in a three-dimensional model of early stage ovarian cancer. Neoplasia. 12, 317-325 (2010).
- Lawrenson, K., Benjamin, E., Turmaine, M. In vitro three-dimensional modelling of human ovarian surface epithelial cells. Cell Prolif. 42, 385-393 (2009).
- Lawrenson, K., Sproul, D., Grun, B. Modelling genetic and clinical heterogeneity in epithelial ovarian cancers. Carcinogenesis. 32, 1540-1549 (2011).
- Grun, B., Benjamin, E., Sinclair, J. Three-dimensional in vitro cell biology models of ovarian and endometrial cancer. Cell Prolif. 42, 219-228 (2009).
- Zietarska, M., Maugard, C. M., Filali-Mouhim, A., Alam-Fahmy, M., Tonin, P. N., Provencher, D. M., Mes-Masson, A. M. Molecular description of a 3D in vitro model for the study of epithelial ovarian cancer (EOC. Mol. Carcinog. 46, 872-885 (2007).
- Shield, K., Ackland, M. L., Ahmed, N., Rice, G. E. Multicellular spheroids in ovarian cancer metastases: Biology and pathology. Gynecol Oncol. 113, 143-148 (2009).
- Dafou, D., Grun, B., Sinclair, J. Microcell-mediated chromosome transfer identifies EPB41L3 as a functional suppressor of epithelial ovarian cancers. Neoplasia. 12, 579-589 (2010).
- Levanon, K., Crum, C., Drapkin, R. New insights into the pathogenesis of serous ovarian cancer and its clinical impact. J. Clin. Oncol. 26, 5284-5293 (2008).
- Kurman, R. J., Shih, I. e. M. Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer–shifting the paradigm. Hum. Pathol. 42, 918-931 (2011).
Tags
Heterotypic Three-dimensional In Vitro ModelingStromal-epithelial InteractionsOvarian Cancer InitiationOvarian Cancer ProgressionEpithelial Ovarian CancersGynecological MalignancySurgical TreatmentsPlatinum-based ChemotherapiesEOC Survival RatesStage I TumorsStage III/IV DiseaseMortality RatesMolecular Genetic BasisBiological BasisEarly Stage Disease DevelopmentMicroenvironment RoleRisk Factors For EOCsNormal Ovary Co-cultureOvarian Surface Epithelial CellsStromal Fibroblast Cells
Citar este artigo
Lawrenson, K., Grun, B., Gayther, S. A. Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression. J. Vis. Exp. (66), e4206, doi:10.3791/4206 (2012).