小鼠外周动脉疾病模型中单核细胞归巢和肿瘤相关血管生成的活体显微镜观察
Summary
单核细胞是外周动脉疾病中 arteriogenesis 的重要介质。本协议采用基底膜样基质和活体显微镜, 研究单核细胞注射在股动脉结扎小鼠模型中的单核细胞归巢和肿瘤相关血管生成。
Abstract
外周动脉疾病和缺血性心脏病的治疗目的是增加血流对血液动力学狭窄引起的缺血性区域。在某些病例中, 血管手术是一个可行的选择, 但是对于没有手术适应症的病人, 如进展到休息疼痛, 严重肢体缺血, 或严重的生命或工作中断, 几乎没有可能减轻他们的疾病。通过刺激侧枝形成的单核细胞强化灌注, 是少数非侵入性选择之一。
我们的小组检查 arteriogenesis 后单核细胞移植到小鼠使用后肢缺血模型。在此之前, 我们已经证明了改进的后肢灌注使用破伤风刺激体单核细胞移植。除了对侧枝形成的影响外, 该疗法还可能影响肿瘤的生长。为了研究这些效应, 我们使用了一个基底膜样基质小鼠模型, 通过注射 Engelbreth-栎-群肉瘤的胞外基质到小鼠的侧面, 在股动脉闭塞后。
在人工肿瘤研究后, 我们使用活体显微镜来研究体内肿瘤血管生成和单核细胞在侧支动脉内的归巢。先前的研究已经描述了动物模型的组织学检查, 这是预料随后对宰后伪影的分析。我们的方法直观地将单核细胞归巢到抵押的实时序列, 易于执行, 并研究了 arteriogenesis 和肿瘤血管生成的过程在体内。
Introduction
心血管疾病, 包括冠心病或周围动脉疾病, 是全球最常见的死亡原因1。细胞疗法是治疗心血管疾病的一种很有前途的方法, 特别是对于那些不能接受手术治疗的人。有几种方法使用细胞或其分泌物质作为治疗工具2,3, 其总体目标是改善缺血性和 underperfused 组织的灌注和维持功能。实现这一目标的一个尝试是改善 arteriogenesis, 从而提高侧枝动脉的发育。单核细胞是一种与抵押相关的重要单元类型。我们的小组专注于研究在炎症区域的单核细胞的影响4,5, 特别是使用后肢缺血模型诱导缺血和随后的炎症6。单核细胞的家到炎症的地区, 并导致复杂的系统性反应, 导致抵押的发展7。
随着活体显微镜的使用, 我们可以研究这些细胞的行为在体内, 并观察被注射的单核生物归巢到炎症区域。大多数以前的研究只描述了验尸分析, 它的缺点包括组织学文物的引入和准备工作所需的大量动物。通过我们的方法, 我们可以通过实时成像在多个时间点来研究免疫过程和侧枝形成。
除了在缺血地区发展侧枝动脉外, 单核细胞还会影响肿瘤的生长。为了研究这些过程, 我们注入了一个基底膜样基质从 Engelbreth-栎-群小鼠肉瘤, 一个富含细胞外基质蛋白的肿瘤8, 并使用活体显微镜进行分析。该矩阵用于筛选血管内皮细胞网络形成或通过血管生成抑制的抗癌治疗的实验分子;在这种情况下, 我们将评估肿瘤血管生成潜能的单核细胞治疗9,10,11。
本议定书的目的是证明一个简单和有效的方法来研究的免疫过程中造成的缺血在一个体内模型。我们可以产生一个更现实的测试环境与组织学检查的验尸肌肉组织。
Protocol
根据德国动物保护法8节的规定, 我们的研究是在萨克森-安哈尔特、Landesverwaltungsamt 哈雷州的许可下进行的。(和 #167; 8, 德国法律的1段为动物保护从 18.05.2016-BGBI。我的 1206, 1313, 和 #167; 31 TierSchVersV 从 13.08.2013). 注意: 在这里的实验中, 使用了8到12周的老雄性 balb/c/c 小鼠, 从献血者的人单核细胞通过活体显微镜将其用于单核细胞的可视化. 1. 单元格准?…
Representative Results
活体显微镜检查肿瘤和侧枝血管的生长触发的单核细胞可以帮助揭示新的方面, 分子机制的肿瘤血管生成和 arteriogenesis。必须使用该协议的步骤精心准备和注射细胞。差异可以导致单一实验之间的变化。单核细胞必须注入静脉系统 (图 1), 以保持系统性的影响, 避免栓子, 这可能发生, 如果注射是在动脉系统进行。 <p class="jove_content" fo:keep-together.within…
Discussion
本文所述方法揭示了侧枝动脉的发展、这些血管中单核细胞的行为以及 arteriogenesis 的过程。应用该协议的步骤很容易学习, 可以用于其他科学领域。尽管有这些优点, 但也有一些缺点。例如, 需要显微设备来执行所描述的技术。为一个实验获取设备是不可持续的, 因此与其他机构合作共享这些设备是很重要的。
与此协议相关的其他困难也可以在实践中避免。一开始, 在显微镜下…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了其他 kr-基金会和 DFG (德意志 Forschungsgemeinschaft, 德国研究基金会) SFB 854 (Sonderforschungsbereich, 合作研究中心) 的支持. 特别感谢汉斯-Holger Gärtner, Audiovisuelles Medienzentrum, 奥托-冯-奥托·冯·格里克大学马格德堡, 马格德堡, 德国, 技术支持。
Materials
| 10% fetal calf serum (FCS) | Sigma Aldrich, Hamburg, Germany | ||
| 1% penicillin/streptomycin | Sigma Aldrich, Hamburg, Germany | ||
| 1mL Omnifix -F insuline syringe | B. Braun, Melsungen AG, Melsungen, Germany | ||
| 50 ml syringe | Fresenius Kabi AG, Bad Homburg, Germany | Injectomat- syringe 50 ml with canule | |
| 6-well-ultra-low-attachement-plates | Corning Incorporated, NY, USA | ||
| 8- 12 week old, male, C57BL/6, BalbC mice | Charles River, Sulzfeld, Germany | ||
| Adhesive tape | TESA SE, Hamburg, Germany | ||
| Acquisition Software | Leica, Wetzlar, Deutschland | Leica Application Suite Advanced Fluorescence (LAS AF); Version: 2.7.3.9723 | |
| Canules | B. Braun, Melsungen AG, Melsungen, Germany | 29G, 30G | |
| Cell culture dish | Greiner Bio-One GmbH, Frickenhausen, Germany | ||
| Cell culture medium | Manufactured by our group with single components | Medium199, 10% Fetal calf serum, 1% Antibiotic (penicillin/streptomycin) | |
| Centrifuge | Beckman Coulter GmbH, Krefeld, Germany | Allegra X-15R centrifuge | |
| Depilatory cream | Veet, Mannheim, Germany | ||
| DiO | Invitrogen Eugene, Oregon, USA | ||
| Disinfection agent | Schülke&Mayr GmbH, Norderstedt, Germany | ||
| Disposable scalpel No.10 | Feather safety razor Co.Ltd, Osaka, Japan | ||
| EDTA | Sigma Aldrich, Hamburg, Germany | ||
| Erlenmeyer flask | GVB, Herzogenrath, Germany | ||
| Ethanol 70% | Otto Fischar GmbH und Co KG, Saarbrücken, Germany | ||
| Fetal Calf Serum | Sigma Aldrich, Hamburg, Germany | ||
| Fine Forceps | Rubis, Stabio, Switzerland | ||
| Flurophor/Rhodamindextran | Thermo Fischer Scientific, Waltham, MA USA | Katalognummer: D-1819 | |
| Gloves | Rösner-Matby Meditrade GmbH, Kiefersfelden, Germany | ||
| Heating pad | Labotect GmbH, Göttingen, Germany | Hot Plate 062 | |
| Human macrophage-colony stimulating factor | Sigma Aldrich, Hamburg, Germany | SRP3110 | |
| Humane leucocyte filters | Blood preservation | ||
| Incubator | Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany | ||
| Isoflurane | Baxter Deutschland GmbH, Unterschleißheim, Germany | ||
| Ketamine (10%) | Ketavet, Pfizer Deutschland GmbH, Berlin , Germany | ||
| Leukocyte separation tubes (tubes with filter) | Bio one GmbH, Frickenhausen, Germany | ||
| Light microscope | Carl Zeiss SMT GmbH, Oberkochen, Germany | Axiovert 40 C | |
| Lymphocyte separation medium LSM1077 | GE Healthcare, Pasching, Austria | ||
| Matrigel | Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA | ||
| Medium M199 | PAA Laboratories GmbH, Pasching, Austria | ||
| Microbiological work bench | Thermo Electron, LED GmbH, Langenselbold, Germany | Hera safe | |
| Microscope slide | Carl Roth GmbH + Co. KG, Karlsruhe | Art. Nr. 1879 | |
| Microscope stand with incubator and heating unit | Leica DMI 6000, Pecon, Germany | ||
| Monocyte wash buffer | Manufactured by our group with single components | PBS, 0,5% BSA, 2mM EDTA | |
| Mouse restrainer | Various | ||
| Multi-photon microscope | Leica, Wetzlar, Deutschland | Leica SP5 Confocal microscope, Cameleon, Coherent | |
| NaCl (0,9%) | Berlin Chemie AG, Berlin, Germany | ||
| Neubauer counting chamber | Paul Marienfeld GmbH und Co.KG, Lauda-Königshofen, Germany | ||
| Objective | Leica, Wetzlar, Deutschland | Leica HC PL APO 10x/0.4 CS | |
| PBS | Life technologies GmbH, Darmstadt, Germany | ph 7,4 sterile | |
| Penicillin/Streptomycin | Sigma Aldrich, Hamburg, Germany | ||
| Percoll | Manufactured by our group with single components | 90 % Percoll, 10% 1,5M NaCl, ρ= 1,064 g cm-3 | |
| Percoll solution | GE Healthcare, Bio-Science AB, Uppsala, Sweden | ||
| Pipettes | Eppendorf AG, Hamburg, Germany | 10µL/100µL/200µL/1000µL | |
| Pipettes serological | Greiner Bio-One GmbH, Frickenhausen, Germany | Cellstar2ml, 5ml, 10ml | |
| Pipetting heads | Eppendorf AG, Hamburg, Germany | ||
| Pipetus | Eppendorf AG, Hamburg, Germany | ||
| Polystyrol tube | Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany | ||
| Scissor | Word Precision Instruments, Inc., Sarasota, USA | ||
| Scale | Mettler PM4800 Delta Range, Mettler-Toledo GmbH, Gießen, Germany | ||
| Suction unit | Integra bioscience, Fernwald, Germany | Vacusafe comfort | |
| Surgical scissors | Word Precision Instruments, Inc., Sarasota, USA | ||
| Trypan blue solution 0,4 % | Sigma Aldrich, Hamburg, Germany | ||
| Tubes with cap | Greiner Bio-One GmbH, Frickenhausen, Germany | 15ml, 50ml Cellstar | |
| Xylazine (2 %) | Ceva Tiergesundheit GmbH, Düsseldorf, Germany |
References
- Volz, K. S., Miljan, E., Khoo, A., Cooke, J. P. Development of pluripotent stem cells for vascular therapy. Vascular pharmacology. 56 (5-6), 288-296 (2012).
- Henry, T. D., et al. The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis. Circulation. 107 (10), 1359-1365 (2003).
- Wagner, M., et al. Isolation and intravenous injection of murine bone marrow derived monocytes. Journal of visualized experiments JoVE. (94), (2014).
- Herold, J., et al. Transplantation of monocytes: a novel strategy for in vivo augmentation of collateral vessel growth. Human gene therapy. 15 (1), 1-12 (2004).
- Ito, W. D., Arras, M., Scholz, D., Winkler, B., Htun, P., Schaper, W. Angiogenesis but not collateral growth is associated with ischemia after femoral artery occlusion. The American journal of physiology. 273 (3 Pt 2), H1255-H1265 (1997).
- Herold, J., et al. Tetanus toxoid-pulsed monocyte vaccination for augmentation of collateral vessel growth. Journal of the American Heart Association. 3 (2), e000611 (2014).
- . Matrigel Matrix Available from: https://www.corning.com/au/en/products/life-sciences/products/surfaces/matrigel-matrix.html (2017)
- Eubank, T. D., Galloway, M., Montague, C. M., Waldman, W. J., Marsh, C. B. M-CSF induces vascular endothelial growth factor production and angiogenic activity from human monocytes. Journal of immunology (Baltimore, Md. 1950). 171 (5), 2637-2643 (2003).
- Fridman, R., et al. Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel. Journal of the National Cancer Institute. 83 (11), 769-774 (1991).
- Woodman, S. E., et al. Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli. The American journal of pathology. 162 (6), 2059-2068 (2003).
- Francke, A., Weinert, S., Strasser, R. H., Braun-Dullaeus, R. C., Herold, J. Transplantation of bone marrow derived monocytes: a novel approach for augmentation of arteriogenesis in a murine model of femoral artery ligation. American journal of translational research. 5 (2), 155-169 (2013).
- Houthuys, E., Movahedi, K., Baetselier, P., de Van Ginderachter, J. o. A., Brouckaert, P. A method for the isolation and purification of mouse peripheral blood monocytes. J. Immunol. Methods. 359 (1-2), 1-10 (2010).
- Berthold, F. Isolation of human monocytes by Ficoll density gradient centrifugation. Blut. 43 (6), 367-371 (1981).
Cite This Article
Wagner, M., Baer, C., Zuschratter, W., Riek-Burchardt, M., Deffge, C., Weinert, S., Lee, J. C., Braun-Dullaeus, R. C., Herold, J. Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease. J. Vis. Exp. (126), e56290, doi:10.3791/56290 (2017).