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A Novel Mouse Skin Graft Model of Vascular Tumors Driven by Akt1
由Akt1驱动的血管肿瘤的新型小鼠皮肤移植模型   

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Cancer Research
Jan 2015

Abstract

To investigate whether endothelial Akt1 activation is sufficient to induce vascular tumor formation in the skin, we have developed a skin graft model in which a skin fragment from transgenic donor mice with inducible and endothelial cell-specific overexpression of activated Akt1 (myrAkt1) is grafted into the skin of wild type recipient mice. The donor skin successfully engrafts after two weeks and, more importantly, vascular tumor develops at the site of transgenic skin graft when myrAkt1 expression is turned on. This skin graft model is a novel approach to investigate the biological impact of a key signal transduction molecule in a temporal, localized and organ-specific manner.

Keywords: Vascular tumors (血管肿瘤), Akt (Akt), Skin graft (皮肤移植), Hemangioma (血管瘤), Animal model (动物模型)

Background

Our research focuses on investigating the role of Akt1 in vascular tumor development. To determine whether the activation of Akt1 in endothelial cells is sufficient to drive de novo vascular tumor formation, we have developed and published a skin graft model of vascular tumor (Phung et al., 2015). We developed the current protocol of grafting skin from transgenic mice onto the skin of wild type mice because this is a way to study the localized and skin-specific effects of the overexpression of constitutively active Akt1 in the vasculature. We have observed that overexpression of Akt1 in the systemic vasculature leads to generalized edema in the lungs and skin, resulting in premature death of transgenic animals within days due to massive pulmonary edema. A procedure to graft transgenic skin onto wild type host mouse allows one to examine the long-term effects of Akt1 overexpression in only the transgenic skin vasculature without the lethality associated with systemic Akt1 overexpression. In our studies, we are particularly interested in investigating the long-term effects of Akt1 in the skin vasculature on the development of vascular tumors based on the hypothesis that hyperactivation of Akt1 in endothelial cells is sufficient to induce vascular tumor formation, thus demonstrating the endothelial cell-autonomous effects of Akt1 in these tumors.

In the model, skin from double transgenic mice with inducible expression of activated Akt1 (myrAkt1) in endothelial cells is grafted onto the back of immunodeficient nu/nu recipient mice. Since myrAkt1 expression is inducible, we can turn off myrAkt1 expression at will by giving the animals tetracycline in their drinking water, and we can turn on myrAkt1 expression by removing tetracycline from the water. A detailed description of the myrAkt1 double transgenic mice has been published (Sun et al., 2005). A schematic of the construction of these mice is shown (Figure 1). Using the skin graft model, we were able to demonstrate that expression of myrAkt1 in endothelial cells is sufficient for the development of vascular tumor. The procedural steps of this model are described below.


Figure 1. myrAkt1 double transgenic mouse model. The VE-Cad:tTA mouse line contains the VE-cadherin promoter cloned upstream of the tetracycline-regulated transcriptional activator (tTA) gene. VE-cadherin promoter is mainly active in endothelial cells. The TET:myrAkt1 line carries a constitutively activated Akt1 with an HA tag (myrAk1) under the control of the tetracycline responsive promoter (TET). To suppress myrAkt1 expression in the double transgenic mice, 1.5 mg/ml tetracycline with 5% sucrose is given to the mice in their drinking water. To turn on myrAkt1 expression, the mice are given pure water without tetracycline. In this system, double transgenic mice express myrAkt1 in endothelial cells, and the expression of myrAkt1 can be regulated with tetracycline (Reference: Sun JF, Phung T, Shiojima I, Felske T, Upalakalin JN, Feng D, Kornaga T, Dor T, Dvorak AM, Walsh K, Benjamin LE. Microvascular patterning is controlled by fine-tuning the Akt signal. Proc Natl Acad Sci USA 2005; 02: 128-33).

Materials and Reagents

  1. Sterile surgical gloves
  2. 10 mm skin punch biopsy instrument (Acu-Punch Biopsy Punch 10 mm) (Acuderm, catalog number: P1025 ) (Figure 2)


    Figure 2. A 10-mm punch biopsy instrument

  3. Ethilon non-absorbable nylon suture, 5-0 PC-3 (Ethicon, catalog number: 1965G )
  4. Sterile surgical towel drape (Steri-Drape) (3M, catalog number: 1010 )
  5. Sterile surgical gauze (4 x 4 Gauze 8 Ply) (COVIDIEN, DermaceaTM, catalog number: 441001 )
  6. Alcohol pads (WebCol Alcohol Prep) (COVIDIEN, catalog number: 6818 )
  7. Donor mice (females, 6-8 weeks old transgenic mice, see reference for the generation of transgenic mice: Sun JF, Phung T, Shiojima I, Felske T, Upalakalin JN, Feng D, Kornaga T, Dor T, Dvorak AM, Walsh K, Benjamin LE. Microvascular patterning is controlled by fine-tuning the Akt signal. Proc Natl Acad Sci U S A 2005; 102: 128-33)
  8. Recipient mice (females, 6-8 weeks old nu/nu mice from Charles River Laboratories, catalog number: 490 )
  9. Isoflurane (Sigma-Aldrich, catalog number: CDS019936 )
  10. Phosphate buffered saline (PBS), 1x sterile solution (AMRESCO, catalog number: K812 )

Equipment

  1. Needle holder (Halsey serrated jaws) (Roboz Surgical Instrument, catalog number: RS-7841 )
  2. Fine single-toothed forceps (Micro dissecting tweezers Pattern 7) (Roboz Surgical Instrument, catalog number: RS-5047 )
  3. Surgical scissors (Micro dissecting scissors) (Roboz Surgical Instrument, catalog number: RS-5910 )
  4. Electric hair shaver (Wahl Battery Operated Compact Travel Trimmer)
  5. Warming plate (Ala Science, catalog number: TCW220x120 )

Procedure

Important: Appropriate Institutional Animal Care and Use Committee (IACCU) approval must be obtained prior to performing the skin graft procedure in animals.

  1. Anesthetize donor and recipient mice with isoflurane anesthetic gas. The donor animal is typically sedated for 5-10 min while the punch biopsy and removal of donor skin is performed. The recipient animal is typically sedated for 20-30 min while the two skin grafts are sutured in place. Animals are adequately sedated when they do not display a response to pinching of the foot pad.
  2. For the donor mouse, chose a skin area from where the skin will be taken. In our study, we selected the back skin because the skin would be grafted at the same anatomic site on the back of the recipient mouse.
  3. Shave the hair over the skin area from where the skin graft will be taken. Be careful to avoid scratching or injuring the skin surface (Figure 3 and Video 1).


    Figure 3. Procedure for removing a punch of skin from donor mouse. A-B. Shave the hair over the skin area from where the skin graft will be taken. C. Hold the skin stretched slightly and position the 10-mm punch instrument vertically on top of the stretched skin. D. Gently rotate the punch instrument until the punch cuts through the skin. E. Once the punch cuts through the skin, remove the punch. F-G. Cut out the piece of punched skin and underlying subcutaneous tissue, and place the skin on a piece of sterile gauze soaked in sterile phosphate buffered saline to keep the skin moist. A close-up photo of the host skin punch with the skin dermis side up is shown in panel G.

    Video 1. Skin graft procedure

  4. Place the animal onto a clean surface covered with a sterile surgical drape.
  5. Clean the shaved skin of the donor animal thoroughly with 70% alcohol wipe pads.
  6. Hold the skin stretched slightly, and position the 10-mm punch instrument vertically on top of the stretched skin. Gently rotate the punch instrument with a smooth 360° turn motion while applying downward pressure until the punch cuts through the skin (also see video demonstration of the technique by Levitt et al., 2013).
  7. Once the punch cuts through the skin and reaches its maximum depth of approximately 1-2 mm through the dermis and subcutaneous tissue, remove the punch. Cut out the piece of punched skin and underlying subcutaneous tissue with sterile fine surgical scissors.
  8. Gently remove the skin fragment with sterile fine forceps. The forceps are sterilized by autoclaving. Please note, when removing the skin, be careful to avoid crushing it by picking up the tissue piece at the edge with fine single-toothed forceps. Save the punched-out skin in order to graft it onto the back of the recipient mouse. Keep the skin graft moist by placing it onto a piece of sterile gauze soaked in 1x sterile phosphate buffered saline (PBS) solution.
  9. Hemostasis can be achieved by applying gentle pressure over the cut area with a dry sterile surgical gauze. Once this step is completed, the donor animal can be immediately euthanized per approved Institutional Animal Care and Use Committee (IACCU) protocol.
  10. Repeat the same punch biopsy procedure for the recipient mouse. Punch out a 10-mm circular piece of skin from the back of the recipient mouse. Cut out the skin fragment and the underlying subcutaneous tissue and discard it (Figure 4 and Video 1).


    Figure 4. Procedure for suturing donor skin onto recipient mouse. A-B. Remove a piece of skin from the back of the recipient mouse using a punch device as described in Figure 3. C. Take the donor skin fragment and put it into place in the punched out area on recipient mouse. D-G. Suture donor skin fragment into place.

  11. Apply gentle pressure over the cut area with a sterile surgical gauze to stop any bleeding.
  12. Take the donor skin fragment and put it into place in the punched out area on the back of the recipient mouse.
  13. To help position the skin graft in place, use the circular skin punch as a clock face. First, place a suture stitch at 12, 3, 6 and 9 o’clock positions. Then add a suture stitch at 1:30, 4:30, 7:30 and 10:30 o’clock positions. Suture the skin fragment in place with non-absorbable nylon suture. Perform double tie knots to secure suture thread in place.
  14. When completed, clean the sutured area by gently wiping it with a sterile gauze soaked in sterile PBS solution.
  15. Keep the animal under observation on a warming plate at 37 °C until it is fully awake and moving, which typically takes about 10-15 min. Animal usually awakens and starts moving soon after anesthesia is withdrawn, so there needs to be constant observation until the animal is fully awake. Once the animal is fully awake and can move, it can be placed back into the cage with clean cage bedding, water and food (place a few food pellets on the cage floor so the animal can feed easily during the first 1-2 days post procedure).
  16. Monitor the animal daily for signs of wound infection and for evidence of healing and wound closure (Figure 5D).
  17. Sutures can be removed 10-14 days after the surgical procedure, which is approximately the time frame needed for complete skin wound healing.
  18. Successful skin engraftment is evident as the grafted donor skin becomes pink with hair growth in the skin graft (Figure 5D).
    Note: Detailed demonstration of the entire procedure can be viewed in our video that is included with this paper. Additional information on how to perform a skin punch biopsy can also be obtained from the video reference in Levitt et al., 2013.

Data analysis

Using this skin graft model to study the effects of endothelial Akt1 on vascular tumor formation, we have found that overexpression of constitutively activated Akt1 for 4 weeks resulted in the development of red masses at the skin graft sites that measured 0.39 ± 0.14 cm3 in size and had histologic features of a vascular tumor (Figures 5 and 6) (also see Figure 3 in Phung et al., 2015). Immunofluorescence staining of tumor sections shows that tumor vessels express the endothelial marker CD31 (Figure 6D). For immunofluorescence staining, fresh tissues are fixed in cold 4% paraformaldehyde, blocked in 5% goat serum for 1 h, and incubated with anti-CD31 antibody (BD Biosciences MEC13.39) overnight at 4 °C, followed by incubation for 1 h with DylightTM 488-conjugated secondary antibody (Jackson ImmunoResearch Labs). More detailed information on the immunofluorescence staining technique and analysis of skin graft tumors is presented in the original publication (adapted from Phung et al., 2015).


Figure 5. Mouse model of endothelial myrAkt1-driven vascular tumor. A. Schematic of myrAkt1 skin graft model of hemangioma. Transgenic donor mice in FVB background express inducible expression of myristoylated activated Akt1 (myrAkt1) in endothelial cells. Skin from myrAkt1 mice (green circles) was used as skin graft donors in nu/nu recipient mice. 10-mm biopsy punches of skin from donor mice were grafted onto the back skin of host (recipient) mice. Grafts were allowed to heal for 2 weeks, at which time half of the recipient mice were taken off tetracycline (given plain drinking water) to turn on myrAkt1 and the other half were kept on tetracycline to turn off myrAkt1 for 4 weeks. B. Photographs of recipient nu/nu mice showing fresh skin grafts with suture on day 0 (arrows). C. Photographs of mice 6 weeks later showing the presence of successful skin engraftment with hair growth in the skin graft (black arrow indicates tuft of white hair in graft site). There is no tumor mass at the skin graft sites in recipient mouse with myrAkt1 OFF, but there are red tumor masses (see red arrow) in the skin graft sites in recipient mouse with myrAkt1 ON. D. Successful skin engraftment is evident by healing of the donor skin graft and the grafted skin becomes pink with hair growth in the graft site (Reference: Phung, T. L., Du, W., Xue, Q., Ayyaswamy, S., Gerald, D., Antonello, Z., Nhek, S., Perruzzi, C. A., Acevedo, I., Ramanna-Valmiki, R., Rodriguez-Waitkus, P., Enayati, L., Hochman, M. L., Lev, D., Geeganage, S. and Benjamin, L. E. (2015). Akt1 and akt3 exert opposing roles in the regulation of vascular tumor growth. Cancer Res 75(1): 40-50).


Figure 6. Endothelial myrAkt1 activation drives vascular tumor formation in vivo. A. Donor mouse skin engrafted in nu/nu recipient mouse as seen by the presence of tuffs of white hair at the graft site. Vascular tumor development in the skin graft was monitored following myrAkt1 induction. Tumor developed in the skin graft 4 weeks following myrAkt1 induction. Magnification X1. B. Microscopic features of the tumor showing a tumor mass (arrows). C. The tumor consists of numerous blood vessels filled with red blood cells in the dermis (arrows). The vessels have variable sizes and irregular lumen with thin vascular wall. D. The tumor is composed of numerous blood vessels as shown in CD31 immunofluorescence stain (green). Cell nuclei are stained with Hoechst dye (blue).

Notes

In our experience, the skin graft procedure works successfully almost all the time as evident by the successful engraftment of the donor skin. We have not observed animal death or skin infection at the graft site. Because immunodeficient nu/nu recipient mice are used as graft recipients, there is no evidence of graft rejection in our study.

Acknowledgments

This work was supported in part by NIH grants R01 CA106263 and P01 CA09264401 (to L. E. Benjamin), American Heart Association 11BGIA5590018, NIH K08 HL087008, NIH R03 AR063223, American Cancer Society 122019-RSG-12-054-01-CSM, and the Baylor Clinical and Translational Research Program (to T. L. Phung).

References

  1. Levitt, J., Bernardo, S., Whang, T. (2013). Videos in clinical medicine. How to perform a punch biopsy of the skin. N Engl J Med 369(11): e13.
  2. Phung, T. L., Du, W., Xue, Q., Ayyaswamy, S., Gerald, D., Antonello, Z., Nhek, S., Perruzzi, C. A., Acevedo, I., Ramanna-Valmiki, R., Rodriguez-Waitkus, P., Enayati, L., Hochman, M. L., Lev, D., Geeganage, S. and Benjamin, L. E. (2015). Akt1 and akt3 exert opposing roles in the regulation of vascular tumor growth. Cancer Res 75(1): 40-50.
  3. Sun, J. F., Phung, T., Shiojima, I., Felske, T., Upalakalin, J. N., Feng, D., Kornaga, T., Dor, T., Dvorak, A.M., Walsh, K. and Benjamin, L. E. (2005). Microvascular patterning is controlled by fine-tuning the Akt signal. Proc Natl Acad Sci U S A 102(1): 128-33.

简介

为了研究内皮Akt1的活化是否足以诱导皮肤血管肿瘤的形成,我们开发出一种皮肤移植模型,其中转基因供体小鼠的皮肤片段具有诱导型和内皮细胞特异性过度表达的活化的Akt1(myrAkt1)被移植入 野生型受体小鼠的皮肤。 供体皮肤在两周后成功植入,更重要的是,当myrAkt1表达被打开时,血管肿瘤发生在转基因皮肤移植物的位点。 这种皮肤移植模型是一种新颖的方法,以时间,局部和器官特异性方式研究关键信号转导分子的生物学影响。
【背景】我们的研究重点是调查Akt1在血管肿瘤发展中的作用。为了确定内皮细胞中Akt1的激活是否足以驱动血管瘤形成,我们开发并发表了一种血管肿瘤的皮肤移植模型(Phung et al。 ,2015)。我们开发了目前将转基因小鼠皮肤移植到野生型小鼠皮肤上的方案,因为这是一种研究组织型活性Akt1在脉管系统中过度表达的局部和皮肤特异性作用的方法。我们已经观察到Akt1在系统性血管系统中的过度表达导致肺和皮肤的广泛性水肿,导致转移性动物在大量肺水肿的几天内过早死亡。将转基因皮肤移植到野生型宿主小鼠上的过程允许人们仅在转基因皮肤脉管系统中检查Akt1过度表达的长期作用,而不与全身性Akt1过表达相关的致死性。在我们的研究中,我们特别感兴趣的是调查Akt1对皮肤脉管系统对血管肿瘤发展的长期影响,这些假说是内皮细胞中Akt1的高活化足以诱导血管肿瘤形成,从而证明内皮细胞Akt1在这些肿瘤中的细胞自主作用。
在模型中,在内皮细胞中具有诱导表达激活的Akt1(myrAkt1)的双转基因小鼠的皮肤移植到免疫缺陷型nu / nu受体小鼠的背部。由于myrAkt1的表达是可诱导的,所以我们可以通过在饮用水中给予动物四环素来关闭myrAkt1表达,我们可以通过从水中除去四环素来打开myrAkt1的表达。已经公开了myrAkt1双转基因小鼠的详细描述(Sun等人,2005)。示出了这些小鼠的构建的示意图(图1)。使用皮肤移植模型,我们能够证明myrAkt1在内皮细胞中的表达对于血管肿瘤的发展是足够的。该模型的程序步骤如下所述。


图1. myrAkt1双转基因小鼠模型。 VE-Cad:tTA小鼠系含有克隆在四环素调节转录激活子(tTA)基因上游的VE-钙粘蛋白启动子。 VE-钙粘蛋白启动子主要在内皮细胞中起作用。 TET:myrAkt1系携带在四环素响应启动子(TET)控制下具有HA标签(myrAk1)的组成型激活的Akt1。为了抑制双转基因小鼠中的myrAkt1表达,在其饮用水中给予1.5mg / ml具有5%蔗糖的四环素的小鼠。为了打开myrAkt1表达,给小鼠给予没有四环素的纯水。在该系统中,双转基因小鼠在内皮细胞中表达myrAkt1,并且可以用四环素调节myrAkt1的表达(参考文献:Sun JF,Phung T,Shiojima I,Felske T,Upalakalin JN,Feng D,Kornaga T,Dor T, Dvorak AM,Walsh K,Benjamin LE.Microvascular patterning is controlled by fine-tuning the Akt signal.Proc Natl Acad Sci USA 2005; 02:128-33)。

关键字:血管肿瘤, Akt, 皮肤移植, 血管瘤, 动物模型

材料和试剂

  1. 无菌手术手套
  2. 10毫米皮肤穿孔活检器械(Acu-Punch活检冲孔10毫米)(Acuderm,目录号:P1025)(图2)


    图2.一个10毫米穿孔活检仪

  3. Ethilon不可吸收尼龙缝线,5-0 PC-3(Ethicon,目录号:1965G)
  4. 无菌手术毛巾悬垂(Steri-drape)(3M,目录号:1010)
  5. 无菌手术纱布(4 x 4 Gauze 8 Ply)(COVIDIEN,Dermacea TM,目录号:441001)
  6. 酒精垫(WebCol Alcohol Prep)(COVIDIEN,目录号:6818)
  7. 331/5000 供体小鼠(雌性,6-8周龄转基因小鼠,参见转基因小鼠的生成:Sun JF,Phung T,Shiojima I,Felske T,Upalakalin JN,Feng D,Kornaga T,Dor T,Dvorak AM,Walsh K,Benjamin LE.Microvascular patterning is controlled by fine-tuning the Akt signal.Proc Natl Acad Sci USA 2005; 102:128-33)
  8. 受体小鼠(雌性,来自Charles River Laboratories的6-8周龄的nu / nu小鼠,目录号:490)
  9. 异氟烷(Sigma-Aldrich,目录号: CDS019936)
  10. 磷酸盐缓冲盐水(PBS),1x无菌溶液(AMRESCO,目录号: K812)

设备

  1. 针座(Halsey锯齿)(Roboz Surgical Instrument,目录号:RS-7841)
  2. 精细单齿镊子(微解剖镊子图案7)(Roboz手术器械,目录号:RS-5047)
  3. 手术剪刀(微解剖剪刀)(Roboz Surgical Instrument,目录号:RS-5910)
  4. 电动剃须刀(Wahl电池操作紧凑型旅行微调器)
  5. 升温板(Ala Science,目录号:TCW220x120)

步骤

重要提示:在进行动物皮肤移植手术之前,必须获得适当的机构动物护理和使用委员会(IACCU)的批准.

  1. 用异氟烷麻醉气体麻醉供体和受体小鼠。 供体动物通常镇静5-10分钟,同时执行穿刺活组织检查和去除供体皮肤。 受体动物通常镇静20-30分钟,同时将两个皮肤移植物缝合到位。 当它们不显示对脚垫的捏合的反应时,动物被充分镇静。
  2. 对于供体老鼠,选择一个皮肤区域,从皮肤将被摄取。 在我们的研究中,我们选择了背部皮肤,因为皮肤将被移植到受体鼠标背部的同一解剖部位。
  3. 将头发刮去皮肤移植物的皮肤区域。 小心避免划伤或伤害皮肤表面(图3和视频1)。


    图3.从供体小鼠中去除皮肤的冲击的步骤。 A-B。 将头发刮去皮肤移植物的皮肤区域。 C.握住皮肤稍微伸展,并将10毫米打孔器垂直放置在拉伸皮肤的顶部。 D.轻轻旋转打孔仪器,直到冲头穿过皮肤。 一旦冲头穿过皮肤,取下冲头。F-G。 切出一块穿孔的皮肤和下面的皮下组织,并将皮肤放在一块无菌纱布上,浸泡在无菌磷酸盐缓冲盐水中以保持皮肤湿润。 皮肤真皮侧面向上的主机皮肤打孔的特写照片如图G所示。

    Video 1. Skin graft procedure

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


  4. 将动物放在用无菌手术罩覆盖的清洁表面上。
  5. 用70%的酒精擦拭垫彻底清洁供体动物的剃光皮肤。
  6. 握住皮肤稍微拉伸,并将10 mm打孔器垂直放置在拉伸皮肤的顶部。轻轻地旋转打孔仪器,平滑360°转动,同时施加向下的压力,直到冲头穿过皮肤(也参见Levitt等人,2013年的技术视频演示)。
  7. 一旦穿孔穿过皮肤并通过真皮和皮下组织达到大约1-2毫米的最大深度,则取下冲头。用无菌的精细手术剪刀剪下一块穿孔的皮肤和下面的皮下组织。
  8. 用无菌细镊子轻轻取出皮肤碎片。镊子通过高压灭菌灭菌。请注意,去除皮肤时,请小心,以避免通过用精细的单齿镊子拾取边缘的组织片来进行粉碎。保存穿孔皮肤,将其嫁接到受体鼠标的背面。通过将皮肤移植物浸泡在浸泡在1x无菌磷酸盐缓冲盐水(PBS)溶液中的无菌纱布上,使皮肤移植物变湿。
  9. 通过用干燥的无菌手术纱布在切割区域上施加温和的压力可以实现止血。一旦这一步骤完成,供体动物可以立即按照经批准的机构动物保护和使用委员会(IACCU)方案进行安乐死。
  10. 对受体鼠标重复相同的穿刺活检程序。从接收鼠标的背面打出10毫米的圆形皮肤。切下皮肤碎片和下面的皮下组织并丢弃(图4和视频1)

    图4.将供体皮肤缝合到受体小鼠上的步骤。 A-B。 使用如图3所示的打孔装置从接收鼠标的背面取下一块皮肤。C.将供体皮肤碎片放入受体鼠标的穿孔区域。D--G。 将供体皮肤片段缝合到位。

  11. 用无菌手术纱布对切割区域施加温和的压力,以止血。
  12. 将供体皮肤碎片放在受体鼠标背部的穿孔区域中。
  13. 为了帮助将皮肤移植物定位到位,请使用圆形皮肤打孔机作为时钟面。首先,在12点,3点,6点和9点钟位置放置缝合针迹。然后在1:30,4:30,7:30和10:30的位置添加缝合线。用不可吸收的尼龙缝合线将皮肤片段缝合到位。执行双重结结,以将缝合线固定到位。
  14. 完成后,用无菌PBS溶液浸泡无菌纱布轻轻擦拭缝合区域。
  15. 将动物在37°C的加温板上观察,直到其完全清醒和移动,通常需要约10-15分钟。动物通常在麻醉撤回后醒来并开始移动,因此需要持续观察,直到动物完全清醒。一旦动物完全清醒并可以移动,它可以放置在笼子里,用干净的笼子寝具,水和食物(在笼子地板上放置几个食物颗粒,使动物可以在头1-2天内轻松饲养程序)。
  16. 每天监测动物的伤口感染迹象,并证明愈合和伤口闭合(图5D)
  17. 缝合线可以在外科手术后10-14天取出,大约是完成皮肤伤口愈合所需的时间。
  18. 成功的皮肤植入是明显的,因为移植的供体皮肤变成粉红色,皮肤移植物中有毛发生长(图5D)。
    注意:可以在本文附带的视频中查看整个过程的详细演示。关于如何进行皮肤穿刺活检的其他信息也可以从Levitt等人,2013年的视频参考中获得。

数据分析

使用这种皮肤移植模型研究内皮Akt1对血管肿瘤形成的影响,我们发现组成型激活的Akt1的过表达4周导致皮肤移植部位的红肿发展,测量为0.39±0.14cm < 3(图5和图6)(参见Phung等人,2015年的图3),其大小具有血管肿瘤的组织学特征。肿瘤切片的免疫荧光染色显示肿瘤血管表达内皮标志物CD31(图6D)。对于免疫荧光染色,将新鲜组织固定在冷的4%多聚甲醛中,在5%山羊血清中封闭1小时,并与抗CD31抗体(BD Biosciences MEC13.39)在4℃下孵育过夜,然后孵育1小时与Dylight 488-缀合的二抗(Jackson ImmunoResearch Labs)。关于免疫荧光染色技术和皮肤移植物肿瘤分析的更详细信息在原始出版物(从Phung等人,2015年改编)中呈现。


图5.内皮myrAkt1驱动的血管肿瘤的小鼠模型。 A.血管瘤myrAkt1皮肤移植模型的示意图。 FVB背景中的转基因供体小鼠在内皮细胞中表达肉豆蔻酰化的活化的Akt1(myrAkt1)的诱导型表达。来自myrAkt1小鼠(绿色圆圈)的皮肤用作nu / nu受体小鼠中的皮肤移植供体。将来自供体小鼠的10-mm活组织检查皮肤移植到宿主(受体)小鼠的背部皮肤上。允许移植物治愈2周,此时一半的受体小鼠被取出四环素(给予普通饮用水)以开启myrAkt1,另一半保留在四环素上以关闭myrAkt1 4周。 B.在第0天(箭头),接受者nu / nu小鼠的照片在缝合处显示新鲜的皮肤移植物。 C.小鼠的照片6周后,皮肤移植物中出现毛发生长成功的皮肤植入(黑色箭头表示移植部位的白色毛发)。在myrAkt1关闭的受体小鼠的皮肤移植部位没有肿瘤块,但在myrAkt1 ON的受体小鼠的皮肤移植部位存在红色肿瘤块(参见红色箭头)。 D.成功的皮肤植入是通过供体皮肤移植物的愈合而显而易见的,并且移植的皮肤在移植部位变成粉红色的毛发生长(参考文献:Phung,TL,Du,W.,Xue,Q.,Ayyaswamy,S.,Gerald ,D.,Antonello,Z.Nhek,S.,Perruzzi,CA,Acevedo,I.,Ramanna-Valmiki,R.,Rodriguez-Waitkus,P.,Enayati,L.,Hochman,ML,Lev,D. ,Geeganage,S。和Benjamin,LE(2015),Akt1和akt3在血管肿瘤生长调节中发挥相反作用。癌症研究75(1):40-50)。


图6.内皮myrAkt1激活驱动体内的血管肿瘤形成。A.在移植部位存在白毛的存在看,在nu / nu受体小鼠中移植的供体小鼠皮肤。 在myrAkt1诱导后监测皮肤移植物中的血管肿瘤发展。 肿瘤在myrAkt1诱导后4周在皮肤移植中发育。 放大倍数X1。 B.显示肿瘤肿块的肿瘤的微观特征(箭头)。 C.肿瘤包括在真皮中充满红细胞的多个血管(箭头)。 血管具有不规则尺寸和不规则管腔,血管壁较薄。 D.肿瘤由多种血管组成,如CD31免疫荧光染色(绿色)所示。 细胞核用Hoechst染料(蓝色)染色。

笔记

在我们的经验中,几乎所有的皮肤移植手术成功地成功地植入了供体皮肤。 我们没有观察到移植部位的动物死亡或皮肤感染。 因为免疫缺陷的nu / nu受体小鼠被用作移植物接受者,我们的研究中没有移植排斥证据。

致谢

这项工作部分由NIH授权R01CA106263和P01CA09264401(LE本杰明),美国心脏协会11BGIA5590018,NIH K08 HL087008,NIH R03 AR063223,美国癌症协会122019-RSG-12-054-01-CSM以及 贝勒临床与转化研究计划(至TL Phung)。

参考

  1. Levitt, J., Bernardo, S., Whang, T. (2013). 临床医学影像。 如何进行皮肤穿孔活检. N Engl J Med 369(11): e13.
  2. Phung, T. L., Du, W., Xue, Q., Ayyaswamy, S., Gerald, D., Antonello, Z., Nhek, S., Perruzzi, C. A., Acevedo, I., Ramanna-Valmiki, R., Rodriguez-Waitkus, P., Enayati, L., Hochman, M. L., Lev, D., Geeganage, S. and Benjamin, L. E. (2015). Akt1和akt3在血管肿瘤生长调节中起反作用. Cancer Res 75(1): 40-50.
  3. Sun, J. F., Phung, T., Shiojima, I., Felske, T., Upalakalin, J. N., Feng, D., Kornaga, T., Dor, T., Dvorak, A.M., Walsh, K. and Benjamin, L. E. (2005). 通过微调Akt信号来控制微血管图案化. Proc Natl Acad Sci U S A 102(1): 128-33.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Phung, T. L. and Ayyaswamy, S. (2017). A Novel Mouse Skin Graft Model of Vascular Tumors Driven by Akt1. Bio-protocol 7(13): e2369. DOI: 10.21769/BioProtoc.2369.
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