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Aorta Ring Assay
主动脉环试验   

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本实验方案简略版
Stem Cells
Jun 2015

Abstract

Angiogenesis is the nature and pathological process of blood vessel growth from pre-existing vascular buds. It plays an important role in cancer and cardiovascular disease. The aorta ring assay is an approach to study angiogenesis. In this experiment, we used the aorta of rat as the study material, cleaned the surrounding tissue of aorta and cut it into 1 mm long rings. Next, the rings were cultured in growth factor-reduced matrigel polymerized at 37 °C. Angiogenesis was assessed at 7 days by using an inverted microscope platform.

Materials and Reagents

  1. 10 cm dish (Thermo Scientific, catalog number: 172931 )
  2. 24 well plates (Thermo Scientific, catalog number: 142475 )
  3. 2 ml and 5 ml disposable sterilized syringe
  4. Sprague-Dawley 4-week old rat (Zhejiang Chinese Medical University Animal Center)
  5. Growth factor-reduced matrigel (Corning, catalog number: 354230 )
  6. Dulbecco’s Modification of Eagle’s Medium with 1 g/L glucose glutamine & sodium pyruvate (Mediatech, catalog number: 10-014-CV )
  7. Fetal Bovine Serum (Biological Industries, catalog number: 04-001-1A )
  8. Phosphate buffer saline (PBS) (Shanghai ji’nuo, catalog number: GNM 20012 )
  9. Chloral hydrate (Guoyao chemical reagent co. LTD, catalog number: 30037517 )
  10. 70% ethanol
  11. Medium (see Recipes)
  12. 4% chloral hydrate (see Recipes)

Equipment

  1. Surgical scissors, scalpel and tweezers
  2. Ruler
  3. 37 °C, 5% CO2 cell culture incubator (Thermo Fisher Scientific, catalog number: 51026334 )
  4. Inverted microscope (Leica Microsystems, model: DMi1 )
  5. Electronic scale

Software

  1. Image-Pro Plus 6.0

Procedure

Note: The whole experiment should be in aseptic conditions including all materials and reagents. All operations should be sterile.

  1. Allow the growth factor-reduced matrigel melt overnight from -20 °C to 4 °C in a refrigerator.
  2. Put 24 well plates and pipette tips in -20 °C chilled overnight.
  3. Use electronic scale to weigh rat.
  4. Use 5 ml disposable sterilized syringe to inject 4% chloral hydrate to rat’s abdomen, 1 ml chloral hydrate per 100 g to anesthesia rat then sacrifice the animal through cervical dislocation, place the rat on the animal operation asepsis.
  5. Spray 70% ethanol to rat’s skin and bundle the rat.
  6. Use scissors to open the chest and remove other organs to expose the Thoracic aorta (Figures 1 and 2).


    Figure 1. Place the rat on the animal operation asepsis


    Figure 2. Open the chest and remove other organs to expose the thoracic aorta

  7. Use tweezers and scissors to separate the spine and aorta (Figure 3).


    Figure 3. Use tweezers and scissors to separate spine and aorta

  8. Cut the aorta from diaphragm to the end of the heart, then put it in 10 cm dish and wash the aorta with sterile PBS at room temperature (Figure 4).


    Figure 4. Cut aorta from diaphragm to the end of the heart

  9. Carefully clean the surrounding tissue around the aorta (Figure 5).


    Figure 5. Clean the surrounding tissue around the aorta

  10. Use scalpel to cut the aorta into sections as 1 mm long rings measured by a ruler (Figure 6).


    Figure 6. Use scalpel to cut aorta into sections as 1 mm long rings measured by a ruler

  11. Keep the growth factor-reduced matrigel on ice. Then add 150 µl matrigel into each well in 24 well plates in biosafety cabinet. Three complex wells for each group.
    Notes:
    1. The matrigel should be careful and quick. It’s easy to become solidified at room temperature.
    2. Keep the matrigel on ice before adding it to the well, but the plate does not need to be put on ice, the plate should be chilled before the matrigel is added.
    3. After adding the matrigel gently shake the plate to be sure the whole surface is covered by matrigel. If there are some bubbles in the well use 2 ml sterilized syringe to break them.
  12. Gently shake the plate then leave it in an ordinary humidified incubator for 30 min (37 °C).
  13. Put the 1 mm aorta ring in the middle of each well.
  14. Then incubate for about 10 min in the ordinary humidified incubator (37 °C).
  15. Add another 150 µl matrigel into the well to cover the ring.
  16. Then incubate for 30 min again in the ordinary humidified incubator (37 °C).
  17. As the aorta ring is embedded in the matrigel then add 200 µl the Dulbecco’s Modification of Eagle’s Medium with 10% Fetal Bovine Serum per well, finally put 24 well plates in the incubator (37 °C) (Figure 7)


    Figure 7. The aorta ring in the matrigel covered by medium in the 24 well plates

  18. Refresh the medium every 2 d.
  19. After 7 d, the branch of vascular is observed under microscope (Figure 8).


    Figure 8. After 7 days, the branch of vascular is observed by microscope. Scale bar, 100 µm.

  20. Use Image-Pro Plus 6.0 to detect the sprouting length and range.
    Total vessel outgrowth is summed for each ring using Image-Pro Plus 6.0 software. The total length of all branches of each ring is calculated so that we can compare them within groups.

Notes

  1. In this protocol we only use one kind of medium (a common medium) to culture.
  2. The students can also collect different kinds of condition media to make more than one group. Usually we set up several groups to compare different supernatants working on the aorta ring.
  3. When you have several groups you need to set up three complex wells for each group.

Recipes

  1. Medium
    90% Dulbecco’s Modification of Eagle’s Medium
    10% Fetal Bovine Serum
  2. 4% chloral hydrate
    0.4 g chloral hydrate
    10 ml deionized water

Acknowledgments

We thank Han et al. for technical advice on Aorta Ring Assay method previously published in Angiogenesis 2012. This work was supported by grants from National Natural Science Foundation of China (No.31171418,81320108003,31371498 for J.W., No.81170308,81370247 for X.Y.H., No.81202948 for L.Z., No.81100141 for J.J.), National High-tech R&D 863 Program (No.2013AA020101) Science and Technology Department of Zhejiang province public welfare projects (No.2013C37054), The National Basic Research Program of China (973Program, No.2014CB965100, 2014CB965103), Major science and technology projects of Zhejiang province (2012C13013-3), National Natural Science Foundation of Chian (No.81573641 for LZ), Zhejiang Provincial Natural Science Foundation (No.LY16H280003 for LZ).

References

  1. Han, Y., Yang, K., Proweller, A., Zhou, G., Jain, M. K. and Ramirez-Bergeron, D. L. (2012). Inhibition of ARNT severely compromises endothelial cell viability and function in response to moderate hypoxia. Angiogenesis 15(3): 409-420.

简介

血管生成是来自预先存在的血管芽的血管生长的性质和病理过程。 它在癌症和心血管疾病中起重要作用。 主动脉环测定是血管生成的一种方法。 在这个实验中,我们使用大鼠主动脉作为材料,清洁主动脉的周围组织,并将其切成1 mm长的环。 然后将环在37℃下聚合的生长因子减少的基质胶中培养。 通过使用倒置显微镜平台在7天评估血管发生。

材料和试剂

  1. 10cm培养皿(Thermo Scientific,目录号:172931)
  2. 24孔板(Thermo Scientific,目录号:142475)
  3. 2 ml和5 ml一次性灭菌注射器
  4. Sprague-Dawley 4周龄大鼠(浙江中医药大学动物中心)
  5. 生长因子减少的基质胶(Corning,目录号:354230)
  6. Dulbecco's Modification of Eagle's Medium with 1g/L glucose glutamine&丙酮酸钠(Mediatech,目录号:10-014-CV)
  7. 胎牛血清(Biological Industries,目录号:04-001-1A)
  8. 磷酸盐缓冲盐水(PBS)(Shanghai ji'nuo,目录号:GNM 20012)
  9. 水合氯化物(国耀化学试剂有限公司,目录号:30037517)
  10. 70%乙醇
  11. 中等(见配方)
  12. 4%水合氯醛(见配方)

设备

  1. 外科剪刀,手术刀和镊子
  2. 标尺
  3. 37℃,5%CO 2细胞培养箱(Thermo Fisher Scientific,目录号:51026334)中。
  4. 倒置显微镜(Leica Microsystems,型号:DMi1)
  5. 电子秤

软件

  1. Image-Pro Plus 6.0

程序

注意:整个实验应在无菌条件下,包括所有材料和试剂。所有操作均应无菌。

  1. 允许生长因子减少基质胶融化过夜从-20°C至4°C在冰箱中。
  2. 将24孔板和移液器吸头在-20°C冷却过夜。
  3. 使用电子秤称重大鼠。
  4. 使用5ml一次性灭菌注射器向大鼠的腹部注射4%水合氯醛,每100克1ml水合氯醛至麻醉大鼠,然后通过颈椎脱位处死动物,将大鼠置于动物手术无菌处。
  5. 喷雾70%乙醇大鼠的皮肤和捆绑的大鼠。
  6. 使用剪刀打开胸部,清除其他器官以暴露胸主动脉(图1和图2)。


    图1.将大鼠置于动物手术无菌状态


    图2.打开胸部并摘除其他器官以暴露胸主动脉

  7. 使用镊子和剪刀分离脊柱和主动脉(图3)。


    图3.使用镊子和剪刀分开脊柱和主动脉

  8. 切开主动脉从膈膜到心脏的末端,然后放在10厘米的菜,用无菌PBS在室温下洗涤主动脉(图4)。


    图4.将主动脉从膈膜切割到心脏末端

  9. 小心清洁周围的组织 主动脉(图5)。

       
    图5.清洁主动脉周围的周围组织

  10. 使用手术刀将主动脉切成由标尺测量的1mm长的环(图6)。

     
    图6.使用手术刀将主动脉切成由标尺
    测量的1 mm长的环
  11. 保持生长因子减少matrigel在冰上。然后在生物安全柜的24孔板中的每个孔中加入150μl基质胶。每组三个复合井。
    注意:
    1. 基质胶应该小心和快速。它很容易在室温下固化。
    2. 将基质胶置于冰上,然后将其加入孔中,但板不需要放在冰上,板应在添加基质胶之前冷冻。
    3. 添加基质胶后轻轻摇动平板以确保整个表面被matrigel覆盖。如果孔中有一些气泡,请使用2 ml灭菌注射器将其破坏。
  12. 轻轻摇动板,然后将其在普通加湿培养箱中30分钟(37℃)。
  13. 将1 mm主动脉环放在每个孔的中间。
  14. 然后在普通加湿培养箱(37℃)中孵育约10分钟。
  15. 添加另外150微升基质胶到孔中以覆盖环。
  16. 然后再在普通加湿培养箱(37℃)中孵育30分钟。
  17. 作为嵌入基质胶中的主动脉环,然后加入200μl含有10%胎牛血清的Dulbecco氏改良Eagle's培养基/孔,最后将24孔板置于培养箱(37℃)(图7)中(图7)

    图7.在24孔板中被培养基覆盖的基质胶中的主动脉环

  18. 每2天更新一次培养基。
  19. 7天后,在显微镜下观察血管分支(图8)

    图8. 7天后,通过显微镜观察血管分支。比例尺,100μm。

  20. 使用Image-Pro Plus 6.0检测出芽长度和范围。
    使用Image-Pro Plus 6.0软件对每个环计算总容器生长。计算每个环的所有分支的总长度,以便我们可以在组内比较它们

笔记

  1. 在这个协议中,我们只使用一种培养基(普通培养基)培养。
  2. 学生还可以收集不同种类的条件媒体来制作多个组。通常我们设置几组,以比较在主动脉环上工作的不同上清液。
  3. 当你有几个组,你需要为每个组设置三个复杂的井

食谱

  1. 中等
    90%Dulbecco's Eagle's Medium修改
    10%胎牛血清
  2. 4%水合氯化
    0.4克水合氯化锌 10ml去离子水

致谢

我们感谢Han em等人。对于先前在"血管生成"2012年发表的主动脉环测定方法的技术建议。这项工作由中国国家自然科学基金会(No.31171418,81320108003,31371498,JW,No.81170308,81370247 XYH,No.81202948浙江省科技厅(No.2013C37054),中国国家基础研究计划(973计划,国家自然科学基金资助项目),国家高技术研究发展计划(No.2013AA020101)浙江省重点科技项目(2012C13013-3),,县国家自然科学基金(浙江省自然科学基金(No.81573641),浙江省自然科学基金(浙江省自然科学基金重点项目)。

参考文献

  1. Han,Y.,Yang,K.,Proweller,A.,Zhou,G.,Jain,MK和Ramirez-Bergeron,DL(2012)。  抑制ARNT严重损害内皮细胞存活力和中度缺氧反应的功能。血管生成 15 (3):409-420。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jin, J., Hu, X., Zhang, L. and Wang, J. (2016). Aorta Ring Assay. Bio-protocol 6(13): e1856. DOI: 10.21769/BioProtoc.1856.
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Dr.Vijay Chidrawar
Northern Border University
Respected Sir, I, Dr. Vijay belong to Department of Pharmacology at Northern Border University, Saudi Arabia. Sir, I'm working on the angiogenesis from last 3-4 years as a part of a funded project. Recently, I came across above procedure of angiogeneis assay by using corning matrigel. The procedure is well explained but I couldn't understand point number 17, and 19. (After 7 d, the branch of vascular is observed under microscope). In the procedure, its stayed to keep well in the incubator at normal temp. but at this temperature, the corning matrigel will solidify, so on the 7th day how the pictures of aorta will be taken? Please clarify my doubt so that I can continue my research. Your help will be highly appreciated. Many Thanks Dr. Vijay
12/13/2017 9:41:46 AM Reply
Jing Jin
Second Affiliated hospital Zhejiang University college of Medicine of clinic research center, China

Corning Matrigel is a solubilized basement membrane, it contains TGF-beta, epidermal growth factor, fibroblast growth factor, tissue plasminogen activator and so on. The matrigel is not strict solidify in 37℃. It imitate the growth environment in the cell, the tissue could be growth in the matrigel

12/26/2017 5:30:54 PM