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Pituitary Isograft Transplantation in Mice
小鼠垂体同系移植物移植   

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参见作者原研究论文

本实验方案简略版
Breast Cancer Research
2 2011

Abstract

The mouse pituitary isograft is a technique developed to administer persistent hormone stimulation, thereby increasing cellular proliferation in the mammary tissue (Christov et al., 1993). The pituitary isograft procedure was first described in ‘Induction of Mammary Cancer in Mice without the Mammary Tumor Agent by Isografts of Hypophyses’ by O. Mühlbock and L. M. Boot in 1959 (Muhlbock and Boot, 1959). Since then, the procedure has seen wide use. A pituitary gland is harvested posthumously from a donor mouse and implanted under the renal capsule of the recipient mouse through a small abdominal incision just below the last rib. Once the pituitary gland is implanted, it begins releasing hormones. These secretions increase serum levels of multiple hormones including prolactin, progesterone and 17β-estradiol (Christov et al., 1993). Although the effects of these hormones on cancer cell proliferation, growth, differentiation, and longevity are not well characterized, and, in some cases, controversial, the net effect of a pituitary isograft is to increase the proliferation of murine breast tissue depending upon strain specific characteristics (Lydon et al., 1999).
   Below is a protocol describing how to perform the pituitary isograft procedure. After many of the steps, a time reference is listed in parentheses. Each reference corresponds to a time point in the embedded video of the procedure. (Video 1)

Video 1. Pituitary isograft transplantation in mice. Video portraying pituitary isograft transplantation procedure in donor and recipient mice.

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Keywords: Pituitary isograft(垂体同系移植物), Mouse model(小鼠模型), Hormone stimulation(激素刺激), Breast cancer(乳腺癌), Cancer model(癌症模型), Pituitary transplantation(垂体移植), Tumor(肿瘤)

Background

Because of the simplicity, longevity, and applicability of the pituitary isograft procedure it has seen substantial and varied usage in the decades since its inception. Uniform, long term hormone stimulation in vivo is difficult to achieve; pituitary isografts deliver this capability. Once implanted pituitary isografts remain effective and safe for the duration of the experimental lifespan.

Materials and Reagents

  1. Surgical gloves (Cardinal Health, catalog number: 2D72N80X )
  2. 1 ml Tuberculin (TB) syringe (BD, catalog number: 309625 )
  3. 18 gauge 1 ½ inch trocar (BD, catalog number: 305196 )
  4. Silk suture 4-0 (Patterson Veterinary Supply, catalog number: 07-891-0230 )
  5. Wound clips (BD, catalog number: 427631 )
  6. Sterile cotton swab (Puritan Medical, catalog number: 25803 2WC )
  7. 8-20-week-old donor and recipient mice of the same strain
  8. Nembutal sodium solution (Pentobarbital sodium injection, USP) (Akorn, catalog number: NDC 76478-501-20 )
  9. Ketoprofen (Patterson Veterinary Supply, catalog number: 07-803-7377 )
  10. Phosphate-buffered saline (PBS) (pH 7.4, 1x, sterile) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  11. 70% ethanol
  12. Antibiotic solution

Equipment

  1. Balance
  2. Sharp-blunt scissors (Fine Science Tool, catalog number: 14028-10 )
  3. Straight flat broad tip forceps (Fisher Scientific, catalog number: 16-100-114 )
  4. Graefe forceps curved serrated (Fine Science Tool, catalog number: 11051-10 )
  5. Graefe forceps straight 1 x 2 teeth (rat tooth) (Fine Science Tool, catalog number: 11053-10 )
  6. Animal clippers (Sunbeam Products, Oster, catalog number: 078005010002 )
  7. Clip applier (BD, catalog number: 427630 )
  8. Clip remover (BD, catalog number: 427637 )

Procedure

  1. Donor mouse surgery: Harvesting the pituitary
    1. Weigh the 8-20-week-old Donor mouse. Donor can be either sex.
    2. Administer 100 mg/kg Nembutal intraperitoneally, a chemical overdose, eventually stopping the mouse’s respiration. Wait until the mouse has no detectable heartbeat then decapitate using sharp-blunt scissors.
    3. Peel the skin off the posterior skull starting from the base of the neck moving anteriorly toward the nose, removing connective tissue as needed. (0:30)
    4. Carefully place the sharp tip of the scissors into the skull of the mouse and cut the skull along the sagittal sutures on either side. (0:40)
    5. Cut along the anterior most suture line connecting your previous two incisions. Then using the straight flat broad tip forceps remove the dome of the skull. (0:48)
    6. Using the straight flat broad tip forceps carefully remove the brain by grasping the parietal lobes and lifting the brain away from the skull. (0:57)
    7. Locate the pituitary gland between the optic nerves and using the curved Graefe forceps outline the pituitary gland to break the membrane holding it in place. Gently lift the pituitary by cradling it between the points of the forceps. (1:04) (see Figure 1)
    8. Place donor pituitary gland into room temperature PBS. (1:25)


      Figure 1. Donor mouse skull with schematic representation showing pituitary gland location. A. Donor mouse skull after brain is removed and the pituitary gland is exposed. B. Donor mouse skull after the brain has been removed with schematic representation overlaid with highlighted regions; base of the cranial cavity (1), Donor mouse pituitary gland (red) (2), donor mouse optic nerves (white) (3).

  2. Recipient mouse surgery: Pituitary isograft
    1. Weigh the 8-20-week-old mouse.
    2. Using 1 ml Tuberculin syringe, administer 50 mg/kg Nembutal as well as 5 mg/kg ketoprofen intraperitoneally. These dosages will provide 1 h of sedation and twenty-four hours of analgesic respectively.
    3. Confirm mouse sedation using a toe pinch to a hind limb. If a reflex is observed wait one minute and attempt again. If after ten minutes a reflex is still observed intraperitoneally administer 12.5 mg/kg Nembutal.
    4. Use clippers to shave the right flank of the mouse from mid ribcage to hip.
    5. Sterilize the abdomen with 70% alcohol. (1:53)
    6. Locate the bottom of the rib cage. Using rat tooth forceps, grasp the skin and lift. Using the pointed end of the sharp-blunt scissors, pierce the tented skin and cut to create a one centimeter incision ending at the bottom most rib. (2:03)
    7. Using rat tooth forceps, grasp the muscles of the abdominal wall exposed by the previous incision and create a smaller incision through the abdominal wall with scissors. (2:17)
    8. Using the eighteen gauge 1.5 inch long beveled barrel trocar draw some PBS into the barrel to moisten. (2:40)
    9. Lay the excised pituitary gland onto the beveled edge of the trocar and draw the pituitary gland entirely into the barrel of the trocar. Place carefully aside, making sure to not dislodge the pituitary gland. (2:47)
    10. Using a sterile cotton swab moistened with PBS, apply pressure ventral to the abdominal incision to expose the kidney through the abdominal wall. Using your thumb and the cotton swab, apply light pressure to keep kidney exposed. (3:25) (see Figure 2)


      Figure 2. Image of recipient mouse right flank. The recipient mouse’s right kidney can be exposed with a cotton swab and finger by using a light pinching pressure applied deep to the ventral and dorsal sides of the highlighted region (1).

    11. While keeping the kidney exposed, use the sharp tip of the curved Graefe forceps to make a 1 mm hole in the renal capsule. Be sure to keep the forceps parallel to the tabletop to avoid damaging the kidney. (3:35)
    12. Insert the trocar bevel edge up through the hole in the renal capsule; again, be careful to keep the trocar parallel to the tabletop to avoid damaging the kidney or tearing the renal capsule. Gently inject the pituitary gland into the capsule. The pituitary should be visible through the capsule. (3:50) (see Figure 3)


      Figure 3. Image depicting correct trocar placement during pituitary gland injection into renal capsule. Notice trocar is parallel with recipient mouse flank and visible through renal capsule. Inset: Schematic representation of proper injection of pituitary gland (1), into exposed renal capsule (2), above the recipient mouse kidney (3).

    13. Using rat tooth forceps lift skin ventral to the incision to place the kidney back into the abdomen. (4:22)
    14. Place three drops of antibiotic solution directly onto the incision site. (4:31)
    15. Sew the abdominal wall incision with two simple interrupted sutures. (4:51)
    16. Close the exterior skin incision with two wound clips. (5:46)

  3. Post-surgery
    1. Place the mouse onto a warm surface. Wait until the righting reflex is restored and the mouse can move about normally. Place mouse back into cage, monitor using regular postoperative criteria.
    2. Intraperitoneally inject ketoprofen 5 mg/kg 24 h later.
    3. Remove wound clips after 7-10 days.

Acknowledgments

This protocol was developed in the Department of Molecular and Cellular Biology, Baylor College of Medicine and Department of Pathology and Laboratory Medicine, University of Kansas Medical School. We thank David Edwards for assistance in the production of the procedure video.

References

  1. Christov, K., Swanson, S. M., Guzman, R. C., Thordarson, G., Jin, E., Talamantes, F. and Nandi, S. (1993). Kinetics of mammary epithelial cell proliferation in pituitary isografted BALB/c mice. Carcinogenesis 14(10): 2019-2025.
  2. Lydon, J. P., Ge, G., Kittrell, F. S., Medina, D. and O'Malley, B. W. (1999). Murine mammary gland carcinogenesis is critically dependent on progesterone receptor function. Cancer Res 59(17): 4276-4284.
  3. Muhlbock, O. and Boot, L. M. (1959). Induction of mammary cancer in mice without the mammary tumor agent by isografts of hypophyses. Cancer Res 19(4): 402-412.

简介

小鼠垂体同种异体移植是开发用于施用持续激素刺激的技术,从而增加乳腺组织中的细胞增殖(Christov等人,1993)。垂体同种异体移植手术首先在O.Mühlbock和L.M. Boot于1959年(Muhlbock和Boot,1959)的“Inction of Mammary Cancer in Mice without the Mammary Tumor Agent by Isoplagen of Hypophyses”中被描述。此后,程序得到广泛的应用。从供体小鼠后期收获垂体腺,并通过位于最后肋骨正下方的小腹切口植入受体小鼠的肾囊下。脑垂体植入后,开始释放激素。这些分泌物增加多种激素的血清水平,包括催乳素,孕酮和17β-雌二醇(Christov等人,1993)。尽管这些激素对癌细胞增殖,生长,分化和寿命的影响没有被很好地表征,并且在一些情况下是有争议的,垂体同种移植物的净效应是根据应变特异性增加鼠乳腺组织的增殖特征(Lydon等人,1999)。
 下面是描述如何进行垂体同种异体移植手术的协议。经过许多步骤,时间参考列在括号中。每个引用对应于该过程的嵌入式视频中的时间点。 (视频1)


背景 由于垂体同种异体移植手术的简单性,寿命和适用性,自成立以来的几十年中已经有了广泛的应用。体内的均匀,长期激素刺激难以实现;垂体同种异体移植能提供此功能。一旦植入垂体同种异体移植物在实验寿命期间保持有效和安全。

关键字:垂体同系移植物, 小鼠模型, 激素刺激, 乳腺癌, 癌症模型, 垂体移植, 肿瘤

材料和试剂

  1. 手术手套(Cardinal Health,目录号:2D72N80X)
  2. 1 ml结核菌素(TB)注射器(BD,目录号:309625)
  3. 18号1½英寸套管针(BD,目录号:305196)
  4. 丝缝4-0(Patterson Veterinary Supply,目录号:07-891-0230)
  5. 伤口夹(BD,目录号:427631)
  6. 无菌棉签(Puritan Medical,目录号:25803 2WC)
  7. 8-20周龄相同菌株的供体和受体小鼠
  8. Nembutal钠溶液(Pentobarbital sodium injection,USP)(Akorn,目录号:NDC 76478-501-20)
  9. 酮洛芬(Patterson Veterinary Supply,目录号:07-803-7377)
  10. 磷酸盐缓冲盐水(PBS)(pH 7.4,1X,无菌)(Thermo Fisher Scientific,Gibco< sup>,目录号:10010023)
  11. 70%乙醇
  12. 抗生素溶液

    设备

    1. 平衡
    2. 锋利的剪刀(精细科学工具,目录号:14028-10)
    3. 直扁平镊子(Fisher Scientific,目录号:16-100-114)
    4. Graefe镊子弯曲锯齿(Fine Science Tool,目录号:11051-10)
    5. Graefe钳直1×2齿(鼠齿)(精细科学工具,目录号:11053-10)
    6. 动物剪刀(Sunbeam Products,Oster,目录号:078005010002)
    7. 夹子施工者(BD,目录号:427630)
    8. 剪辑删除器(BD,目录号:427637)

      程序

      1. 供体小鼠手术:收获垂体
        1. 称重8-20周龄的Donor鼠标。捐助者可以是性别。
        2. 腹膜内施用100mg / kg Nembutal,化学物质过量,最终阻止老鼠的呼吸。等到鼠标没有可检测到的心跳,然后用锋利的剪刀剪断。
        3. 从颈部的基部开始从后方颅骨剥离皮肤,向前移动到鼻子,根据需要去除结缔组织。 (0:30)
        4. 小心地将剪刀的锋利尖端放在鼠标的头骨中,并沿着两侧的矢状缝线切割头骨。 (0:40)
        5. 沿着连接您以前的两个切口的最前线缝线切开。然后使用直的扁平的尖端镊子去除颅骨的圆顶。 (0:48)
        6. 使用直的扁平尖端镊子通过抓住顶叶并将脑部从头骨上抬起来小心地移除大脑。 (0:57)
        7. 找到视神经之间的垂体,并使用弯曲的Graefe镊子轮廓垂体以将其保持在适当位置。通过在镊子的点之间摇摆来轻轻地抬起垂体。 (1:04)(见图1)
        8. 将供体垂体腺置于室温PBS中。 (1:25)


          图1.供体小鼠头骨,其示意图显示垂体腺位置。 A.取出大脑后的供体小鼠头骨,暴露垂体腺。 B.大脑已经被去除后的供体小鼠头骨,其示意性表示与突出区域重叠;颅底(1),供体小鼠垂体腺(红色)(2),供体小鼠视神经(白色)(3))。

        9. 受体小鼠手术:垂体同种移植物
          1. 称重8-20周龄的老鼠。
          2. 使用1ml结核菌素注射器,腹膜内给予50mg / kg Nembutal以及5mg / kg酮洛芬。这些剂量分别提供1小时的镇静和二十四小时的镇痛药
          3. 使用脚趾捏到后肢确认小鼠镇静。如果观察到反射等待一分钟再次尝试。如果十分钟后仍然观察到反射,腹膜内给药12.5mg / kg Nembutal。
          4. 使用剪刀从中间胸部到臀部刮鼠标的右侧。
          5. 用70%酒精消毒腹部。 (1:53)
          6. 找到肋骨的底部。使用鼠齿镊子,抓住皮肤和抬起。使用锋利的剪刀的尖端,刺破帐篷皮肤并切割,以创建一个厘米切口,最终处于最底部的肋骨。 (2:03)
          7. 使用大鼠牙镊子,抓住先前切口暴露的腹壁肌肉,并用剪刀通过腹壁创建较小的切口。 (2:17)
          8. 使用十八米1.5英寸长的斜面桶套管针将一些PBS吸入桶中以润湿。 (2:40)
          9. 将切除的垂体腺置于套管针的斜边上,将垂体腺体完全吸入套管针筒内。仔细放在一边,确保不会脱落垂体。 (2:47)
          10. 使用用PBS浸湿的无菌棉签,将腹部压腹压至腹部切口,使肾穿过腹壁。使用拇指和棉签,施加轻微的压力以保持肾脏暴露。 (3:25)(见图2)


            图2.受体小鼠右侧腹部的图像通过使用深度施加到突出显示区域的腹侧和背侧的轻微的夹紧压力,可以用棉签和手指将受体鼠的右肾暴露( 1)
          11. 在保持肾脏暴露的同时,使用弯曲的Graefe镊子的锋利尖端在肾囊中形成1mm孔。确保将镊子平行于桌面,以免损坏肾脏。 (3:35)
          12. 将套管针斜角边缘向上插入肾囊中的孔;再次,小心保持套管针平行于桌面,以避免损坏肾脏或撕开肾囊。轻轻地将垂体注入胶囊内。垂体应通过胶囊可见。 (3:50)(见图3)


            图3.垂体腺注射入肾囊内的正确套管针放置的图像。注意:套管针与受体小鼠侧腹平行,通过肾囊可见。插图:正确注射垂体腺(1),暴露于肾囊(2),受体小鼠肾脏上方(3)的示意图。

          13. 使用鼠齿镊子将皮肤腹部提升至切口,将肾脏放回腹部。 (4:22)
          14. 将三滴抗生素溶液直接放入切口部位。 (4:31)
          15. 用两个简单的中断缝线缝腹壁切口。 (4:51)
          16. 用两个伤口夹闭合外部皮肤切口。 (5:46)

          17. 手术后
            1. 将鼠标放在温暖的表面上。等到正确的反射恢复,鼠标可以正常移动。将鼠标放回笼中,使用常规术后标准进行监测。
            2. 24小时后腹腔注射酮洛芬5 mg / kg
            3. 7-10天后取出伤口夹

              致谢

              该协议是在堪萨斯大学医学院贝勒医学院和病理学和实验室医学系的分子和细胞生物学系开发的。我们感谢David Edwards在制作程序视频方面提供协助。

              参考

              1. Christov,K.,Swanson,SM,Guzman,RC,Thordarson,G.,Jin,E.,Talamantes,F.and Nandi,S。(1993)。垂体同种移植的BALB / c小鼠中乳腺上皮细胞增殖的动力学。致癌作用 14(10):2019-2025。
              2. Lydon,JP,Ge,G.,Kittrell,FS,Medina,D.and O'Malley,BW(1999)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm .nih.gov / pubmed / 10485472“target =”_ blank“>小鼠乳腺癌发生严重依赖于孕酮受体功能。 59(17):4276-4284。 br />
              3. Muhlbock,O. and Boot,LM(1959)。  通过垂体的同种移植诱导乳腺癌的乳腺癌。 19(4):402-412。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Walker, C., Hong, Y., Kittrell, F., Medina, D., Edwards, D. and Behbod, F. (2017). Pituitary Isograft Transplantation in Mice. Bio-protocol 7(11): e2317. DOI: 10.21769/BioProtoc.2317.
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