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In vivo Chick Chorioallantoic Membrane (CAM) Angiogenesis Assays
雏鸡绒毛尿囊膜(CAM)体内血管形成实验   

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Abstract

Angiogenesis is the process of formation of new blood vessels from pre-existing vessels or endothelial cell progenitors. It plays a crucial role in tumor growth and metastasis. Tumor angiogenesis have been widely studied as an important target for suppressing tumor growth and metastasis. Here, we describe an in vivo chick embryo chorioallantoic membrane (CAM) model. The chick embryo chorioallantoic membrane is an extraembryonic and is rich of blood vessels. After exposing the vascular zone of the CAM, a sterilized filter-paper disk is employed, which is used as a carrier for being loaded with various chemicals, drugs or virus. Finally, the CAM was fixed and spread on glass slide, and the blood vessels were quantified by counting the number of blood vessel branch points. Compared with the matrigel plug angiogenesis assay, in which tumor cells are mixed with the matrigel gel (expensive) and injected into the mice, subsequently using immunohistochemistry (IHC) staining (time consuming) with the endothelial marker to indicate the presence of the newly formed capillaries, the main advantages of CAM model are its low cost, simplicity, reproducibility, and reliability. Thus, the CAM can be widely used in vivo to study both angiogenesis and anti-angiogenesis.

Keywords: Chick chorioallantoic membrane(鸡胚绒毛尿囊膜), Angiogenesis(血管生成), Blood vessel branch(血管分支)

Materials and Reagents

  1. Fertilized E6 chicken embryos (from Poultry Center of South China Agricultural University)
  2. 0.1% Benzalkonium Bromide (from Guangzhou Chemical Reagent Factory, diluted in sterile water before use)
  3. Methanol and acetone (1:1 in volume)
  4. Filter-paper disk (Whatman, catalog number: 1441150 )
  5. Packing film (Parafilm)

Equipment

  1. Incubator
  2. Glass slide
  3. Ophthalmic forceps

Procedure

  1. Fertilized E6 chicken embryos (48 ± 5 g) were cleaned with 0.1% Benzalkonium Bromide and preincubated at 37.5 °C in 85% humidity for 2 days.
  2. Egg morphology appears like a meta-ellipse, with a relatively larger side and a smaller one, and the air sac is usually located on the larger side right behind the shell. After disinfection of the shell center outside the air sac with 0.1% Benzalkonium Bromide, a hole highlighted with marker pen was buffed and drilled gently over the air sac with a nipper not to break the shell, and the vascular zone was easy to be identified on the CAM (Figure 1).


    Figure 1. The vascular zone of the CAM

  3. Two drops of normal saline were then added to moisten the inner shell membrane adjacent to the CAM so that the membrane was easy to be separated from CAM.
  4. After being clamped and raised by ophthalmic forceps, the membrane and the CAM separated unforcedly, and then a 1 x 1 cm window on the membrane was sectioned to expose the vascular zone.
  5. A 5 mm x 5 mm sterilized filter-paper disks, which were used as a carrier for being directly loaded with indicated concentrations of chemicals or virus, were then directly applied and adhere to the vascular zone with right density of vascular.
  6. Upon sealing the openings with sterile flexible packing film, the eggs were further incubated for indicated periods.
  7. Finally, a mix of methanol and acetone (1:1 in volume) was directly added to immerse and fix the blood vessels of the experiment zone.
  8. After being clamped and raised by ophthalmic forceps, the CAM was easy to be separated from the embryo, and it was cut and spread on glass slide, and the blood vessels were viewed, photographed and quantified by counting the number of blood vessel branch points (Figure 2).


    Figure 2. Blood vessel branch points on CAM. Arrow indicates new-formed blood vessel branches.

Acknowledgments

This protocol was adapted from the following published papers: Wen et al. (2013); Chen et al. (2014). This work was supported by The State Key Development Program for Basic Research of China (2009CB 918904, 2013CB945203), National Natural Sciences Foundation of China (30870955, 91029727, 30900555) and Program for New Century Excellent Talents in University (NCET-08-0646).

References

  1. Chen, Z., Zhang, Y., Jia, C., Wang, Y., Lai, P., Zhou, X., Wang, Y., Song, Q., Lin, J., Ren, Z., Gao, Q., Zhao, Z., Zheng, H., Wan, Z., Gao, T., Zhao, A., Dai, Y. and Bai, X. (2014). mTORC1/2 targeted by n-3 polyunsaturated fatty acids in the prevention of mammary tumorigenesis and tumor progression. Oncogene 33(37): 4548-4557.
  2. Wen, Z. H., Su, Y. C., Lai, P. L., Zhang, Y., Xu, Y. F., Zhao, A., Yao, G. Y., Jia, C. H., Lin, J., Xu, S., Wang, L., Wang, X. K., Liu, A. L., Jiang, Y., Dai, Y. F. and Bai, X. C. (2013). Critical role of arachidonic acid-activated mTOR signaling in breast carcinogenesis and angiogenesis. Oncogene 32(2): 160-170.

简介

血管生成是从预先存在的血管或内皮细胞祖细胞形成新血管的过程。它在肿瘤生长和转移中起关键作用。肿瘤血管生成已被广泛研究为抑制肿瘤生长和转移的重要靶标。在这里,我们描述了一种体内小鸡胚胎绒毛膜尿囊膜(CAM)模型。小鸡胚胎绒毛尿囊膜是一种胚外和富含血管。在暴露CAM的血管区后,使用灭菌的滤纸盘,其用作用于装载各种化学品,药物或病毒的载体。最后,将CAM固定并铺展在载玻片上,通过计数血管分支点的数量来量化血管。与基质胶塞血管生成测定相比,其中肿瘤细胞与基质胶凝胶(昂贵)混合并注射到小鼠中,随后使用内皮标记的免疫组织化学(IHC)染色(耗时)来指示新形成的毛细管,CAM模型的主要优点是其低成本,简单,可重复性和可靠性。因此,CAM可以在体内广泛使用以研究血管生成和抗血管生成。

关键字:鸡胚绒毛尿囊膜, 血管生成, 血管分支

材料和试剂

  1. 受精E6鸡胚(来自华南农业大学家禽中心)
  2. 0.1%苯扎氯铵(来自广州化学试剂厂,在使用前用无菌水稀释)
  3. 甲醇和丙酮(体积比为1:1)
  4. 滤纸盘(Whatman,目录号:1441150)
  5. 包装膜(Parafilm)

设备

  1. 孵化器
  2. 玻璃片
  3. 眼科钳

程序

  1. 用0.1%苯扎氯铵清洁受精的E6鸡胚(48±5g),并在37.5℃,85%湿度下预培养2天。
  2. 蛋形态看起来像一个元椭圆,具有相对较大的边和较小的一边,气囊通常位于壳体后面的较大一侧。用0.1%苯扎氯铵消毒空气囊外壳中心,用标记笔突出的孔用空气囊轻轻打磨,用钳子不破坏壳,血管区容易鉴定CAM(图1)。


    图1. CAM的血管区

  3. 然后加入两滴生理盐水以润湿邻近CAM的内壳膜,使得膜容易与CAM分离。
  4. 在被眼科钳夹住和抬起后,膜和CAM未被强制分离,然后将膜上的1×1cm窗口切开以暴露血管区。
  5. 然后直接施加5mm×5mm灭菌的滤纸盘,其用作直接装载有指定浓度的化学品或病毒的载体,并且粘附到血管区 右密度的血管。
  6. 在用无菌软包装膜密封开口时,将鸡蛋进一步温育指定的时间。
  7. 最后,直接加入甲醇和丙酮(体积比为1:1)的混合物,以浸没和固定实验区的血管。
  8. 在用眼科钳夹住和抬起后,CAM容易从胚胎中分离,并且将其切割并铺展在载玻片上,通过计数血管分支点的数量来观察,拍照和定量血管 图2)。


    图2. CAM上的血管分支点。箭头表示新形成的血管分支。

致谢

该协议改编自以下发表的论文:Wen等人(2013); Chen (2014)。这项工作得到了国家基础研究国家重点发展计划(2009CB 918904,2013CB945203),中国国家自然科学基金(30870955,91029727,30900555)和大学新世纪优秀人才计划(NCET-08-0646 )。

参考文献

  1. Chen,Z.,Zhang,Y.,Jia,C.,Wang,Y.,Lai,P.,Zhou,X.,Wang,Y.,Song,Q.,Lin,J.,Ren,高,Q,赵,Z.,郑,H.,万,Z.,高,T.,赵,A.,戴,Y.和白,X。(2014)。 mTORC1/2被n-3多不饱和脂肪酸靶向以预防乳腺肿瘤发生和肿瘤进展。 Oncogene 33(37):4548-4557
  2. 温,ZH,苏,YC,莱,PL,张,Y,徐,YF,赵,A.,姚,GY,贾,CH,林,J.,徐, ,XK,Liu,AL,Jiang,Y.,Dai,YF和Bai,XC(2013)。 花生四烯酸活化的mTOR信号在乳腺癌发生和血管生成中的关键作用 Oncogene 32(2):160-170。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Z., Wen, Z. and Bai, X. (2013). In vivo Chick Chorioallantoic Membrane (CAM) Angiogenesis Assays. Bio-protocol 3(18): e913. DOI: 10.21769/BioProtoc.913.
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surendra reddy
yogivemanauniversity
sir . please tell me the controls(positve and negetive) which you are used and what concentrations are used
7/6/2016 12:34:03 AM Reply
Zhenguo Chen
Department of Cell Biology, Southern Medical University, China

Thanks for your interest. Could you please specify for what positive and negative controls you mentioned.

7/10/2016 7:40:45 PM


surendra reddy
yogivemanauniversity

for CAM i have taken suramin disodium salt as controller . please tell me the other controllers

7/13/2016 10:53:39 PM


Zhenguo Chen
Department of Cell Biology, Southern Medical University, China

You use suramin disodium as a control for what chemicals?

7/13/2016 11:16:09 PM


surendra reddy
yogivemanauniversity

phycocyanin

7/13/2016 11:38:27 PM


Zhenguo Chen
Department of Cell Biology, Southern Medical University, China

But we did not mention phycocyanin in the protocol.

7/14/2016 12:08:51 AM


Hruaia Pautu
Entomology Research Institute
Firstly, I've been looking for CAM assay, most of them did not mention details protocol.
Thanks for your article.
Secondly, For cleaning purpose besides 0.1% Benzalkonium Bromide, what else can we use?
2/9/2016 9:47:56 AM Reply
Zhenguo Chen
Department of Cell Biology, Southern Medical University, China

To our knownledge, neither 75%alcohol nor iodophor was suitable, so 0.1% Benzalkonium Bromide is the best choice. Thanks for your inquiry。

7/10/2016 7:31:59 PM


glad mohesh
SSSMCRI
dear author.your article is informative, however as a new aspirant to learn this technique kindly help me with your pdf or video files.thankyou
4/8/2015 9:13:22 AM Reply
Zhenguo Chen
Department of Cell Biology, Southern Medical University, China

A PDF can be downloaded directly from the website.Thank you for your interest.

4/15/2015 7:36:15 PM