癌症转移，使用禽流感胚胎模型的定量分析
Summary
使用定量PCR技术，我们展示了如何建立鸡胚CAM模型可用于定量分析的人类肿瘤细胞转移到远处器官。
Abstract
在转移的癌细胞从原发肿瘤的传播，侵入到周围组织，并扩散到远处器官。转移是一个复杂的过程，可能涉及到许多的组织类型，变量跨度的时间段，经常发生深刻的内部器官，使其难以进行调查和量化。此外，转移过程中的疗效的影响使其难以评估单一的肿瘤细胞行为方面的贡献，在转移级联的多个步骤。因此，经常进行转移检测在实验动物提供了一定的现实背景下，在其中学习转移。不幸的是，这些模型进一步复杂，其复杂的生理。鸡胚是一个独特的体内模型，克服了很多的局限性研究转移，由于绒毛尿囊膜（CAM），一个良好的血管多余的胚胎组织位于下面的蛋壳，是接受了肿瘤细胞的xenografting无障碍（图1）。此外，由于鸡胚是自然免疫缺陷，CAM容易支持正常和肿瘤组织植入。最重要的是，禽流感的CAM成功地支持大多数癌症细胞的特点，包括生长，侵袭，血管生成，和微环境重塑。这使得模型特别有用的调查导致癌转移的途径，并预测转移性癌的新的潜在疗法的反应。物种的具体铝PCR检测细胞播散，使人们有可能定量评估，是殖民统治只有25细胞的转移器官。使用人类的表皮细胞株（HEp3），我们使用这个模型来分析癌细胞的自发转移到远处器官，包括鸡胚肝脏和肺。此外，我们使用ALU – PCR协议表明，作为一种工具来分析和定量转移的单个元素的血管内，逮捕，外渗，和殖民的灵敏度和可重复性的检测。
Protocol
1。准备xenografting鸡蛋（自发转移） 刚下岗的受精鸡蛋孵化在一个旋转的孵化器，在100 ° F和60％的湿度为10天。鸡蛋旋转四次，每一个小时。 发育10天的鸡蛋都放在一个鸡蛋机架就在自己身边。一个鹅颈灯或其他合适的光源是用蜡烛鸡蛋的蛋壳射入airsac是位于蛋的钝端的光。 位于绒毛尿囊静脉和标记。此静脉是位于顶部的蛋壳是几个大血管交界。在此分支点血管下降，离从CA…
Discussion
鸡胚CAM已使用了几十年，作为一个模型系统，研究各方面的癌症生物学和转移性进展。原则上转移能力的评估已经在这个模型中进行的各种团体，包括休眠分析（阿吉雷Ghiso等，1999; Ossowski和帝国，1983年），组织重塑 （Deryugina等，2005; 。哈恩Dantona 等 ，1999），和转移（泽吉尔斯达等 ，2008;泽吉尔斯达等 ，2002）。它仍然是一个非常有吸引力的癌症生物学的，因为?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要认识到泰森食品公司慷慨地提供受精卵。莎娜阿诺德在本议定书的拍摄。帕尔默Trenis露丝属Kirschstein国家研究服务奖（F31的美国国家癌症研究所）是由美国国立卫生研究院的支持下。部分支持这项工作是由CCSRI格兰特＃700537 JDL和NIH / NCI授予＃CA120711 – 01A1和CA120711 – 01A1对AZ。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| 1X Dulbecco’s Modified Eagle Medium (DMEM) | Invitrogen | 11995073 | |
| Dulbecco’s Phosphate-Buffered Saline (D-PBS) (1X), liquid | Invitrogen | 14190250 | pH 7.4 |
| Trypsin, 0.05% (1X) with EDTA 4Na, liquid | Invitrogen | 25300054 | |
| Sportsman hatcher | Berry Hill | 1550HA | These incubators are some of the most readily available. However, the video will show different equipment. |
| Sportsman incubator | Berry Hill | 1502EA | |
| Dremel rotary tool | Dremel | ||
| Dremel cutoff wheels no. 36 | Dremel | 409 | |
| Portable Pipette Aid | Fisher | 1368115E | |
| Cotton Tipped Applicators | Fisher | 23-400-001) | |
| Fertilized Eggs | Various | Many different vendor can be used. The eggs should be approximately 35-55 grams. The hens that lays them should be between 36 and 55 weeks of age. | |
| Hemocytometer | Hausser Scientific, VWR | 15170-090 | |
| Fiber-optic microscope illuminator | Amscope | HL250-AY | 150W |
| 25 gauge needle | |||
| Sybr Green Extract–N-Amp Tissue PCR Kit | Sigma-Aldrich | XNATRG |
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Citazione di questo articolo
Palmer, T. D., Lewis, J., Zijlstra, A. Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model. J. Vis. Exp. (51), e2815, doi:10.3791/2815 (2011).