小鼠中Pax8定向缺失肿瘤抑制因子 Arid1a 和 Pten 的子宫内膜癌进展的非侵入性超声评估
1Department of Molecular and Comparative Pathobiology,Johns Hopkins University School of Medicine, 2Department of Obstetrics and Gynecology,Johns Hopkins University School of Medicine, 3Texas Tech University School of Veterinary Medicine, 4Department of Pathology,Johns Hopkins University School of Medicine, 5Department of Surgery,Johns Hopkins University School of Medicine, 6Department of Coloproctological Surgery,Juntendo University School of Medicine, 7Department of Human Pathology,Juntendo University School of Medicine, 8Sidney Kimmel Comprehensive Cancer Center,Johns Hopkins University
Summary
该协议描述了一种使用与大体和组织学变化相关的超声成像在子宫内膜癌诱导小鼠模型中监测子宫中形态变化随时间进展的方法。
Abstract
由于这些模型中易于处理和遗传操作，可以在小鼠中研究子宫癌。然而，这些研究通常仅限于评估在不同队列的多个时间点安乐死的动物的病理学死后，这增加了研究所需的小鼠数量。纵向研究中的小鼠成像可以跟踪个体动物的疾病进展，减少所需的小鼠数量。超声技术的进步使得检测组织中的微米级变化成为可能。超声已被用于研究卵巢中的卵泡成熟和异种移植物的生长，但尚未应用于小鼠子宫的形态变化。该协议检查了诱导子宫内膜癌小鼠模型中病理学与 体内 成像比较的并置。超声观察到的特征与大体病理学和组织学观察到的变化程度一致。发现超声波对观察到的病理具有高度预测性，支持将超声检查纳入子宫疾病（如小鼠癌症）的纵向研究中。
Introduction
小鼠仍然是生殖疾病最重要的动物模型之一1，2，3。卵巢癌和子宫癌有几种转基因或诱导的啮齿动物模型。这些研究通常依赖于在不同时间点安乐死的多个队列来捕捉形态和病理变化的纵向趋势。这阻止了在单个小鼠中获取有关癌症发展的连续数据的能力。此外，在不知道个体小鼠疾病进展状态的情况下，干预研究基于预定的时间点和先前队列的平均结果，而不是检测特定动物进展的个体阈值4，5。因此，需要允许对活体动物进行纵向评估的成像方法，以促进用于测试新药或化合物的临床前模型，并加速对病理生物学的理解，同时提高严谨性和可重复性6。
超声成像（US）是纵向监测小鼠子宫癌进展的一种有吸引力的方法，因为与其他成像方法相比，它相对简单且便宜，易于执行，并且可以具有显着的分辨率6，7。这种非侵入性方式可以在清醒小鼠或短暂镇静下的小鼠中使用5-10分钟的检查捕获微米级的特征。超声显微镜已被验证为测量小鼠卵巢卵泡发育8和植入或诱导的肿瘤生长的方法9，10，11。高频US也被用于经皮宫内注射12和观察大鼠子宫在发情周期的变化13。高频US可用于固定在专用固定平台上的小鼠，使用导轨系统固定换能器/探头，以捕获具有标准化位置和压力的高分辨率图像;但是，并非所有机构都提供此设备。手持式换能器扫描方法可以采用较少的专用设备，并用于小鼠的临床诊断和研究应用。
问题仍然是，使用手持式高频探头进行美国成像是否可以用于监测数周的子宫癌发展。与肠道类似，啮齿动物子宫是一种薄壁细长的结构，在腹部内非常移动，并且通过多个组织深度连续，这使得成像比相对不动的器官（如肾脏）更具挑战性。本研究旨在建立超声观察到的组织与组织病理学之间的相关性，确定定位小鼠子宫的标志，并确定子宫内膜癌纵向评估的可行性。这项研究提供的数据显示了US成像的子宫外观与组织病理学之间的定性对应关系，以及小鼠在几周内的连续成像。这些结果表明，手持式US可用于监测小鼠子宫内膜癌的发展，从而为收集单个小鼠纵向数据以研究子宫癌创造了机会，而无需专用设备。
Protocol
所有涉及小鼠的程序和实验均按照约翰霍普金斯大学动物护理和使用委员会批准的方案进行。对于所有程序，都穿着适当的个人防护装备，包括手套和一次性隔离衣。处理锐器时采取了预防措施，使用后立即妥善处理在红箱锐器容器中。有关本协议中使用的所有材料和设备的详细信息，请参阅 材料表 。 1.用多西环素诱导iPAD小鼠（诱导 Pten，Arid1a 双缺?…
Representative Results
Pax8-Cre-Arid1a-Pten 双缺失 （iPAD） 转基因小鼠维持在混合遗传背景 （129S， BALB/C， C57BL/6） 上，如前所述14.小鼠均饲喂多西环素饲料2周以诱导Cre重组酶。在我们小组以前的工作中，多西环素通过管饲法给药14;然而，在目前的研究中，多西环素饲料诱导方法有效地工作并降低了小鼠的管饲压力。重要的是要检查多西环素给药方法是否足以诱导Cre重组酶…
Discussion
该协议检查了超声在评估小鼠子宫腺癌进展中的子宫形态变化的效用。在这项研究中，通过纵向跟踪小鼠子宫内膜癌的诱导，发现超声检测到的解剖细节是大体和组织病理学的指标。这为使用纵向研究打开了大门，在多个时间点通过超声监测较少数量的小鼠来跟踪小鼠子宫癌的进展。这种纵向检测是通过使用手持式探头完成的，无需使用轨道系统超声设备。使用的高频探头（换能器）广泛使用，?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢NCI卵巢癌孢子计划P50CA228991，博士后培训计划5T32OD011089和约翰霍普金斯大学Richard W. TeLinde捐赠基金的资助。该项目的部分资金还来自日本私立学校促进和互助公司对私立高等教育机构的经常性支出补贴。
Materials
| Reagents and Equipment Used for Animal Care | |||
| Rodent Diet (2018, 625 Doxycycline) | Envigio | TD.01306 | Mouse Feed |
| Reagents and Equipment Used for Ultrasound Imaging | |||
| 10 mL injectable 0.9% NaCl | Hospira, Inc | RL-7302 | Isotonic Fluid |
| Absorbent Pad with Plastic Backing | Daigger | EF8313 | Absorbant Pads |
| Anesthesia Induction Chambers | Harvard Apparatus | 75-2029 | Induction Chamber |
| Anesthetic absorber kit with absorber canister, holder, tubing, & adapters | CWE, Inc | 13-20000 | Nose Cone and Tubing |
| Aquasonic Clear Ultrasound Gel (0.25 Liter) | Parker Laboratoies | 08-03 | Ultrasound Gel |
| BD Plastipak 3 mL Syringe | BD Biosciences | 309657 | Syringe |
| F/Air Scavenger Charcoal Canister | OMNICON | 80120 | Scavenging System for Anesthesia |
| Isoflurane, USP | Vet One | 502017 | Anesthesia Agent |
| M1050 Non-Rebreathing Mobile Anesthesia Machine | Scivena Scientific | M1050 | Anestheic Vaporizer |
| MX550S, 25-55 MHz Transducer, 15mm, Linear | VisualSonics | MX550S | Ultrasound Transducer (Probe) |
| Nair Hair Aloe & Lanolin Hair Removal Lotion – 9.0 oz | Nair | Depilliating Cream | |
| Philips Norelco Multigroomer All-in-One Trimmer Series 7000 | Philips North America | MG7750 | Clippers |
| PrecisionGlide 25 G 1" Needle | BD Biosciences | 305125 | Needle |
| Puralube Ophthalmic Ointment | Dechra | 17033-211-38 | Lubricating Eye Drops |
| Vevo 3100 Imaging System | VisualSonics | Vevo 3100 | Ultrasound Machine |
| Vevo LAB 5.6.1 | VisualSonics | Vevo LAB 5.6.1 | Ultrasound Analysis Software |
| Vinyl Heating Pad with cover, 12 x 15" | Sunbeam | 731-500-000R | Heating Pad |
| Wd Elements 2TB Basic Storage | Western Digital Elements | WDBU6Y0020BBK-WESN | Data Storage |
| Reagents and Equipment Used for Immunohistochemistry | |||
| 10% w/v Formalin | Fischer Scientific | SF98-4 | Tissue Fixation Buffer |
| Animal-Free Blocker and Diluent, R.T.U. | Vector Laboratories Inc. | SP5035 | Antibody Blocker |
| Charged Super Frost Plus Glass Slides | VWR | 4831-703 | Tissue Mounting Slides |
| Citrate Buffer | MilliporeSigma | C9999-1000ML | Epitope Retrival Buffer (pTEN) |
| Cytoseal – 60 | Thermo Scientific | 8310-4 | Resin for Slide Sealing |
| Gold Seal Cover Glass | Thermo Scientific | 3322 | Coverslide |
| Harris Modified Hematoxylin | MilliporeSigma | HHS32-1L | Counterstain Buffer |
| Hybridization Incubator (Dual Chamber) | Fischer Scientific | 13-247-30Q | Oven to Melt Parraffin |
| ImmPACT DAB Substrate, Peroxidase (HRP) | Vector Laboratories Inc. | SK-4105 | Signal Development Substrate |
| ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase | Vector Laboratories Inc. | MP-7451 | Secondary IHC Antibody |
| Oster 5712 Digital Food Steamer | Oster | 5712 | Vegetable Steamer for Epitope Retrival |
| rabbit mAB anti-ARID1a | abcam | ab182560 | Primary IHC Antibody (1:1,000) |
| rabbit mAB anti-PTEN | Cell Signaling | 9559 | Primary IHC Antibody (1:100) |
| Scotts Tap Water Substitute | MilliporeSigma | S5134-100ML | "Blueing" Buffer |
| Tissue Path IV Cassette | Fischer Scientific | 22272416 | Tissue Fixation Cassette |
| Trilogy Buffer | Cell Marque | 920P-10 | Epitope Retrival Buffer (ARID1a) |
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Diesen Artikel zitieren
Vistein, R., Winer, B., Myers, S., Liberto, J., Ishiyama, S., Guo, X., Saeki, H., Wang, T., Shih, I., Gabrielson, K. Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice. J. Vis. Exp. (192), e64732, doi:10.3791/64732 (2023).