鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性
Summary
在这里，我们提出了一个协议入侵肿瘤细胞研究到生活的正常组织碎片在三个方面。这种器官培养技术主要应用于体外测试潜在抗侵袭药物。
Abstract
小鸡心脏检测的目标是提供一个相关的器官培养的方法来研究肿瘤浸润在三个维度。该分析方法可侵入性和非侵入性的细胞之间的区别，并允许试验化合物对肿瘤侵袭的影响研究。癌细胞 – 无论是作为聚集体或单个细胞 – 都面临着小鸡胚胎心脏的片段。在悬浮器官培养几天或几周后，将面对培养物固定并包埋在石蜡中用于组织学分析。癌细胞和正常组织之间的三维相互作用，然后从用苏木精 – 曙红或免疫组化染色的表位在心脏组织或对抗癌细胞后的连续切片重建。该测定是用最近的概念，癌浸润是癌细胞和它们相邻的基质主元件（肌成纤维细胞，endoth之间的分子相互作用的结果是一致的elial细胞，细胞外基质成分等）。在这里，这种基质环境提供给癌细胞作为活体组织片段。配套方面来测定的相关性是多方面的。侵袭测定中是按照癌症侵袭的标准：在时间渐进占领和更换和宿主组织的空间，以及侵袭和非侵袭性的对抗细胞体内通常与该测定的结果。此外，细胞在体内的侵入图案，由病理学家所定义，是反映在组织学图像中测定。定量结构活性关系（QSAR）分析与众多潜在抗侵入有机同类化合物所得到的结果所允许的在临床中使用的类黄酮和查耳酮，和已知的抗转移药的结构-活性关系（ 例如，微管抑制剂的研究）抑制侵袭测定中也是如此。 HoweveR，该法没有考虑到癌症侵袭账户免疫学的贡献。
Introduction
侵袭是恶性肿瘤的标志。此活动不仅导致周围组织的破坏，但也牵连转移形成。由于癌症患者从浸润和转移，以及高效抗侵入性治疗死仍然很少，实验室化验模仿的肿瘤细胞的侵袭已经开发出来。小鸡心脏检测的目标是提供一个相关的器官培养的方法来研究肿瘤浸润在三个维度。该分析方法可侵入性和非侵入性的细胞之间的区别，并允许研究试验化合物对肿瘤的侵袭的影响。
使用该测定的背后的基本原理是实际的概念，即肿瘤是生态系统，其中的肿瘤细胞不断地与他们的基质（宿主细胞和细胞外基质）相互作用，并通过这些分子间的相互作用的入侵是微调1。所以，在测定肿瘤细胞所面临的LIVI纳克胚胎鸡心脏片段2，它不仅作为由肿瘤细胞侵袭基板，还可以作为不同类型的基质细胞和矩阵元素的来源。小鸡心脏包含肌细胞，成纤维细胞和内皮细胞，以及细胞外基质由层粘连蛋白，纤连蛋白和不同类型的胶原蛋白的。以这种方式，三维器官培养技术涵盖牵连侵袭患者肿瘤的许多细胞和分子的相互作用。
小鸡心脏测定的主要优点是对基质的实施效果。这方面比体外其他入侵检测是基于肿瘤细胞侵入到非生物凝胶基底膜3或间质基质4分子构成更加完整。在器官培养实验发现肿瘤细胞和正常的生活宿主组织之间对抗的概念已经推出几个作者包括沃尔夫和施奈德在法国5，Easty和Easty在英国6和施莱希德国7。以上所引用的方法的鸡心脏侵袭测定两种技术优点是，片段的体积很容易被标准化，并且它们保持收缩，这允许功能完整性监视器官培养期间。此外，禽类胚胎是优选的，因为它们可以容易地从蛋的无菌内容解剖。该测定具有通过提供周围的肿瘤细胞的复合基质概念相似小鸡尿囊绒膜膜试验8。
该测定法已成功应用于从同一人肿瘤侵入性和非侵入性的细胞的变体之间进行区分，例如，在MCF-7（乳房）9和HCT-8（结肠）10细胞系的家庭。该技术可用于检测潜在的抗侵入性化合物以及<suP> 11,12。如进一步说明的，它可以用于建立有机小分子的结构 – 活性关系。该试验确实，但是，没有考虑到免疫细胞对癌症侵袭的贡献。应当强调，该技术不能被认为是因为高数操作的，试验运行的有限数目（最多30培养物）和长的周转时间（约1个月），为高通量分析系统。
Protocol
Representative Results
Discussion
期间的PHF的制备，所述片段可以不留在悬浮液中，但附着于血管壁;这可以通过增加培养基的体积来克服。如果的PHF的数目太低，其大小过大，降低培养基的体积。测试电池的故障聚集，可能是由于在该温度或微生物感染的波动。可替代地，一个无法聚集可能是该细胞的固有特性。期间附着聚集到PHF的，附着力差可以由延伸在半固体琼脂培养基的顶部的潜伏期或通过吸收滤纸装置除去周围的培养多…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Marleen De Meulemeester for demonstrating the assay technique in the video film. B. I. R. is a Postdoctoral Research Fellow of the Research Foundation – Flanders (FWO – Vlaanderen). L.M.M. is a recipient of an Emmanuel van der Schueren grant from the Flemish League against Cancer (Vlaamse Liga tegen Kanker).
Materials
| Ringer's salt solution | Braun | CEO123 | |
| Bacto-agar | Becton Dickinson | 214010 | |
| Tris-(hydroxymethyl)-aminomethaan Analar | VWR | 103157P | |
| BSA : albumin from bovine serum, cohn V fract. | Sigma | A4503-500G | |
| MEM-Rega 3 | Life Technologies | 19993013 | |
| Gyrotory shakers | New Brunswick Scientific | G10 and G33 | |
| Erlenmeyer flasks 50 ml | Novolab | 92717 | |
| Glass Petri dishes | Novolab | 68516 | |
| Iridectomy scissors | Rumex | 11-0625 | |
| Macroscope with calibrated ocular grid | Wild | 157702 | |
| Paraffin wax | International Medical products | 8599956 | |
| Eosin Y | Sigma | 230251-25g | |
| Harris' hematoxylin | Sigma | HHS32-1L | |
| Coverslipping resin (Tissue-Tek) | Sakura | 4494 | |
| Paraffin melting apparatus | GFL | 1052 | |
| Microtome for paraffin sectioning | Reichert | ||
| Stppers for Erlemeyer flasks with gas in- and outlets | Novolab | 2602421 and 260243 | |
| Diaminobenzidine | Sigma | D8001 | |
| REAX rocking table | Heidolph | 54131 | |
| 24-well tissue dishes | VWR | NUNC142475 | |
| Ophtalmological enucleation spoon | Rumex | 16-060 | |
| Sharp forceps | Rumex | 4-111T | |
| Blunt forceps | Nickel-Electro LTD | 7112 | |
| Erlenmeyer flasks 5 ml | Novolab | 211901 | |
| Microscope slides washed and degreased | International Medical products | 8613908 |
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