小鸡前OVO文化前OVO CAM含量：它是如何真正起作用
Summary
鸡胚绒毛尿囊膜（CAM）是一个独特的，自然免疫缺陷的支持文化环境研究血管生成和肿瘤发生。此视频文章演示了在鸡的不同步骤<em>前OVO</em>企业文化，潜在的血管生成的物质和成功接种肿瘤细胞和组织上的CAM表面的应用程序。
Abstract
在繁殖初期的鸡蛋之间在体外和体内系统，并提供了血管的测试环境，不仅要研究血管生成，但更要研究肿瘤的发生。后的鸡胚绒毛尿囊膜（CAM）的发展，其血管网络可以轻松地访问，操纵和观察，因此对血管生成的实验提供了一个最佳设置。由于淋巴系统没有完全开发的后期阶段，直到孵化，鸡胚作为一个能够维持无特定物种的限制嫁接的组织和细胞的自然免疫缺陷的主机。除了培育发展异体和异种移植，CAM血管网络提供了一个独特的支持性环境对肿瘤细胞的血管内，传播和血管逮捕和被逮捕细胞渗出，形成微转移灶的一个仓库里。
实验目的，在最近期的研究CAM暴露切割通过蛋壳的一个窗口，并进行了实验在 OVO无障碍CAM和效果的观察和照片文件的可能性，造成很大的局限性。当鸡胚无壳培养使用1-4，提供了无实验细节，如果在所有出版，这些文化的存活率低。我们精制的鸡胚前OVO文化的方法显着引进的鸡蛋含量的合理控制挤压。这些当然OVO文化，加强无障碍CAM和鸡胚，使容易在体内影响的文件，并促进胚胎的实验操作。这使得大量的科学问题进行了成功的应用：（1）作为改善血管生成的实验5,6，（2）实验设立便利注射在鸡胚眼7-9的玻璃体，（3）作为一个测试环境中传播和血管内的分散维持移植成功和肢芽鸡和小鼠13以及文化作为一种改进系统 肿瘤细胞从细胞接种于CAM 10-12线，（4（5）嫁接，繁殖，重新嫁接坚实的原发肿瘤组织活检获得表面上的CAM 14）为。
在这个视频文章中，我们描述了建立一个精致的小鸡前OVO文化和CAM与存活率的检测，50％以上。除了 我们提供了一个一步一步前OVO文化的科学应用的大量成功应用的示范。
丹尼尔Dohle，苏珊D.婆娑，和塞巴斯蒂安Gustmann同等贡献这项研究。
Protocol
所有的设备和试剂必须购买无菌或需要加热或蒸汽消毒或用70％乙醇消毒。 作者状态，对动物的实验是根据欧洲共同体理事会指令（86/609/EEC），美国国立卫生研究院的机构动物所规定的实验程序和法规的动物护理和使用准则保养和使用委员会（IACUC）在杜伊斯堡 – 埃森大学（德国）。 第1部分：蛋孵化受精卵可存储长达一个星期在13 ° C。 鸡…
Discussion
创新或只是另一个小鸡文化协议？
1-4与前壳的文化协议鸡胚的总生存率分别为低，例如CA。 30％1。此视频文章中所述的成品前OVO文化协议，相反，允许超过50％的生存率。 OVO文化经典相比前鸡胚的 OVO文化，大大方便了CAM和鸡胚的无障碍，使他们的实验操作和连续监测。这种文化的方法，可用于各种广泛的应用，增强血管生成</s…
Acknowledgements
作者想感谢优秀的技术援助和教授Ruebben J. Kueper J. Wittschier慷慨地提供肿瘤物质。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Fertilized Eggs | Animal | Söries Trockels, Germany | ||
| Mice | Animal | Charles Rivers Laboratories | ||
| Incubator Type 3000 with rotating egg tray | Tools | Siepmann GmbH Germany | 9503 | Maintain at 37°C with relative humidity set above 60% |
| Egg incubator BSS 160 | Tools | Grumbach, Germany | 8101 | Maintain at 37-387deg;C with relative humidity set above 60% |
| PBS | Reagent | Sigma | PBS should be cold (> 4°C) and sterile | |
| Dulbecco’s modified eagle’s medium | Reagent | Sigma | D 6429 | DMEM culture medium |
| Leibovitz’s L-15 Medium | Reagent | Invitrogen | 31415-029 | here: collection medium for tumor samples |
| Tumor cell -specific medium | Reagent | Sigma | each research group should use the medium they culture their tumors cells in or common culture media, e.g. Leibovitz’s Medium or Dulbecco’s modified eagle’s medium supplemented with 15% fetal bovine serum (FBS), 4mM L-glutamine and 50 μM insulin |
|
| 70% EtOH | Reagent | Sigma | ||
| Magnetic stir bar, triangled 80 mm | Tool | VWR | 442-0391 | A triangled magnetic stir bar serves well to crack the eggs shell. |
| Forceps DUMONT #5 | Tool | Fine Science Tools | 11252-30 | bevelled very fine shanks (0.05 mm x 0.02 mm tip) |
| Forceps DUMONT #7 | Tool | Fine Science Tools | 11271-30 | curved shanks (0.07 mm x 0.10 mm tip) |
| Spring scissors, straight, 8cm | Tool | Fine Science Tools | 15000-00 | fine, small straight blades |
| Fine Iris scissors, straight | Tool | Fine Science Tools | 14094-11 | Use to cut our the CAM |
| Standard scissors, straight, sharp/blunt | Tool | Fine Science Tools | 14007-14 | Use for decapitation or cervical dislocation |
| microliter syringe 1702 TLLX, 25 μl | Tool | Hamilton | 80222 | Use for injection into the vitreous |
| Hamilton 33-G needle (15 mm) | Tool | Fine Science Tools | 18073-15 | Use for injection into the vitreous |
| Sterile scalpels | Tool | Use for dissecting tumor samples | ||
| Small drain spoon | Tool | Geuder, Germany | 15758 | Use to transfer of small chick embryos |
| Wax board with fixing pins | Tool | Self made | Use to fix of animals for dissection | |
| Petri dishes 60 x 15 mm | Tool | Greiner | 628102 | |
| Petri dishes 92 x 16 mm | Tool | Sarstedt | 82.1472 | |
| Sterile Petri dish 100 x 20 mm | Tool | Greiner | 664160 | extra deep, with spacers for ventilation |
| Beaker | Tool | 300-600 ml | ||
| FALCON tubes | Tool | FALCON | 15 ml and 50 ml | |
| Eppendorf tubes | Tool | Eppendorf | 1.5 ml and 2 ml | |
| 1ml pipette tips | Tool | Use to cut plastic rings for application of substances / cells | ||
| Peripheral venous catheter (PVL) | Tool | Viggo® | Use to cut silicon rings from the tube for application of substances / cells | |
| Pipettes and tips | Tool | Eppendorf / Abimed | ||
| Autoclaved folded Filters 595 1/2, 110 mm diameter | Tool | Schleicher & Schuell | 311643 | Carrier, which caused least irritation of the CAM; used in the paper |
| PTFE-Pledget, non resorbing, 6,3 x 152.4 x 1.6 mm | Tool | santec-medical | REF 277, LOT 832/511-1 | Carrier; caused false positive results |
| Hxdroxyethylcellulose Tylose H 100000 | Reagent | Carrier mixed with Kollidon 17 PF; caused false positive results | ||
| Kollidon 17 PF | Reagent | BASF | S30200 | mixed with Hydroxyethylcellulose; caused false positive results |
| Collagen Biomatrix TissuDura | Tool | Baxter Healthcare S.A. | REF B2246000999999 | Carrier; caused false positive results |
| Round glass cover slips, 11 mm | Tool | Assistent Germany | 1001/0011 | Carrier; causes false positive results |
| Bovine Collagen Sponge | Tool | Wyeth | Carrier; caused false positive results | |
| Resodont Absorbable Collagen Membrane | Tool | Resorba Woundcare Germany | LOT 280303 | Carrier; caused false positive results |
| Non-Woven Swabs | Tool | Fink & Walter GmbH | REF 328132 | Carrier; caused false positive results |
Referências
- Auerbach, R., Kubai, L., Knighton, D., Folkman, J. A simple procedure for long-term cultivation of chicken embryos. Dev. Biol. 41, 391-394 (1974).
- Tufan, A. C., Akdogan, I., Adiguzel, E. Shell-less culture of the chick embryo as a model system in the study of development neurobiology. Neuroanatomy. 3, 8-11 (2004).
- Deryugina, E. I., Quigley, J. P. Chick embryo chorioallantoic membrane models to quantify angiogenesis induced by inflammatory and tumor cells or purified effector molecules. Methods Enzymology. 444, 21-44 (2008).
- Ribatti, D. Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis. Int. Rev. Cell Mol. Biol. 270, 181-224 (2008).
- Höckel, M., Sasse, J., Wissler, J. H. Purified monocyte-derived angiogenic substance (angiotropin) stimulates migration, phenotypic changes, and “tube formation” but not proliferation of capillary endothelial cells in vitro. J. Cell Physiol. 133, 1-13 (1987).
- Wissler, J. H. Engineering of blood vessel patterns by Angio Morphogens [Angiotropins]. Materialwiss. Werkstofftech. 32, 984-1008 (2001).
- Kistler, H. B. . J. r. .., LaVail, J. H. Penetration of horseradish peroxidase into the optic nerve after vitreal or vascular injections in the developing chick. Invest Ophthalmol. Vis. Sci. 20, 705-716 (1981).
- Halfter, W. Change in embryonic eye size and retinal cell proliferation following intravitreal injection of glycosaminoglycans. Invest. Ophthalmol. Vis. Sci. 49, 3289-3298 (2008).
- Oppitz, M., Busch, C., Shriek, G., Metzger, M., Just, L., Drews, U. Non-malignant migration of B16 mouse melanoma cells in the neural crest and invasive growth in the eye cup of the chick embryo. Melanoma Res. 17, 17-30 (2007).
- Uchibayashi, T., Egawa, M., Nakajima, K., Hisazumi, H., Tanaka, M., Endo, Y., Sasaki, T. Responses of tumour cell lines implanted onto the chorioallantoic membrane of chick embryo to anticancer agents in combination with hyperthermia. Urol. Res. 20, 237-239 (1992).
- Demir, R., Naschberger, L., Demir, I., Melling, N., Dimmler, A., Papadopoulus, T., Sturzl, M., Klein, P., Hohenberger, W. Hypoxia generates a more invasive phenotype of tumour cells: an in vivo experimental setup based on the chorioallantoic membrane. Pathol. Oncol. Res. , (2008).
- Kunzi-Rapp, K., Genze, F., Kufer, R., Reich, E., Hautmann, R. E., Gschwend, J. E. Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model. J. Urol. 166, 1502-1507 (2001).
- Hall, B. K. Grafting of organs and tissues to the chorioallantoic membrane of the embryonic chick. TCA Manual. 4 (3), 881-883 (1978).
- Kunzi-Rapp, K., Kaskel, P., Steiner, R., Peter, R. U., Krahn, G. Increased blood levels of human S100 in melanoma chick embryo xenografts’ circulation. Pigment Cell Res. 14, 9-13 (2001).
- McFall, R. C., Sery, T. W., Makadon, M. Characterization of a new continuous cell line derived from a human retinoblastoma. Pesquisa do Câncer. 37, 1003-1010 (1977).
- Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. P. Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination. Pesquisa do Câncer. 65, 10959-10969 (2005).
- Kim, J., Kovalski, K., Ossowski, L. requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell. 94, 353-362 (1998).
Citar este artigo
Dohle, D. S., Pasa, S. D., Gustmann, S., Laub, M., Wissler, J. H., Jennissen, H. P., Dünker, N. Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works. J. Vis. Exp. (33), e1620, doi:10.3791/1620 (2009).