大鼠乳腺内皮细胞移植到间膜白脂肪垫
Summary
本文介绍了一种移植方法，将供体大鼠乳腺上皮细胞移植到受体动物的细胞间白脂肪垫中。该方法可用于检查宿主和/或供体对乳腺上皮发育的影响，并消除预清除的需要，从而扩展了此技术的实用性。
Abstract
早在20世纪70年代，研究人员就成功地将乳腺上皮细胞移植到大鼠的间膜白色脂肪垫中。使用移植技术移植乳腺上皮，利用青春期啮齿动物宿主提供的荷尔蒙环境。这些研究非常适合探索各种生物操作对乳腺发育的影响，并剖析乳腺生物学的许多方面。一个常见但有限的特征是，移植的上皮细胞受到周围频闪的强烈影响，并且被内源上皮竞争;为了利用本地乳腺组织，必须在移植前清除腹腔白色脂肪垫以去除宿主乳腺上皮。使用大鼠模型生物体的一个主要障碍是，清除在绝育后大鼠中发育的乳腺树是无效的。当移植到无腺脂肪垫中时，供体上皮细胞可以重新填充清除的宿主脂肪垫，形成功能性乳腺。隔膜脂肪垫是这些移植物的替代位置。一个主要的优势是，它缺乏导管结构，但提供了正常的频闪，这是促进上皮外生长所必需的，很容易在大鼠中访问。这种技术的另一个主要优点是，它是微创的，因为它消除了烧灼和去除生长的内源性乳腺树的需要。此外，隔膜脂肪垫包含一个中血管，可用于分离移植部位。由于内源性腺体保持不变，该技术也可用于将内源性乳腺与移植腺体进行比较的研究。本文介绍了乳腺上皮细胞移植到大鼠的间膜白脂肪垫中的方法。
Introduction
产后乳腺发育和导管形态形成是青春期开始时主要受荷尔蒙信号影响的过程。在小鼠和大鼠中，通常使用的乳腺生物学模型生物，这个过程开始于大约3周的年龄，快速增殖和分化导致成熟乳腺的形成。成熟的乳腺可以经历无数次的扩张和进化，自20世纪初以来，这种财产一直在被调查。在高增增和癌症发展的背景下，乳腺移植技术在20世纪50年代发展，并在1977年古尔德等人贡献的定量方法增强。啮齿动物移植技术的完善，有助于在理解正常乳腺生物学方面取得重大进展，这些生物学仍被广泛用于研究各种治疗和基因操作对正常乳腺发育和疾病状态的影响。
许多假设已经产生，随后测试使用乳腺移植，第一次由DeOme等人描述在1959年1。数十年的实验表明，从供体乳腺切除的导管组织倾向于重新填充整个脂肪垫5，6，7，并表明乳腺发育的一个关键成分存在于这些上皮结构。后来对小鼠的研究表明，单个乳腺干细胞可以重新填充一个清除的脂肪垫，并有助于发现一个单一的，共同的祖细胞的基础和发光乳腺上皮细胞8，9，10。根据这些结论，有人建议移植增加具有多系再生潜力的细胞池，由于可塑性，使移植细胞生长功能性乳腺7，10，11，12，13。重要的是，在啮齿动物中使用移植技术克服了细胞培养引起的异常的局限性14，而且往往在短短几周内就产生结果。
虽然这个程序最初是在小鼠前体病变的背景下描述的，但很快又扩展到了大鼠，并结合致癌物治疗，建立了多种性，作为癌症易感性的衡量标准，但移植技术的普及伴随着每个物种遗传工具的发展。虽然结合移植的小鼠研究提供了许多转化性发现，但大鼠乳腺的乳腺瘤与人类更相似，与16、17号相似，为研究雌激素受体阳性（ER+）乳腺癌提供了明显的优势。乳腺肿瘤在这两个物种中都具有诱导性，但它们在激素敏感性和基因表达谱方面有所不同。主要区别是大鼠乳腺肿瘤表达和依赖于卵巢和垂体激素受体的功能，即雌激素和孕激素 （PR），类似于人类乳腺癌的 Luminal-A 亚型。事实上，乳腺上皮细胞移植，如本议定书所述，已用于研究涉及乳腺癌的基因变异，并确定细胞自主性对乳腺上皮细胞的影响18。
除了肿瘤生物学，正常大鼠乳腺的导管上皮表现出更高的分枝水平，并侧翼比小鼠更厚的频闪层。在乳腺上皮移植研究中，频闪的重要性得到了很好的记录。乳腺上皮必须与脂肪性血质相互作用，理想情况下，它自己的美感，以经历其特有的形态形成19，20。将组织移植到受体乳腺中提供最佳环境;然而，内源上皮的存在会干扰结果。预清除内源上皮的乳腺通常在小鼠移植测定中进行，需要手术切除内源性乳腺组织和/或切除奶嘴1、21、22。虽然有可能，预清除绝育后大鼠的乳腺上皮并不广泛，主要是因为在戒除后大鼠中清除生长的乳腺树无效。由于已经表明，在身体的其他部位的脂肪组织区域可以支持移植的乳腺上皮21、23、24的生长，因此通过在体间白色脂肪垫中移植组织，在大鼠中很容易避免预清除的过程。
本文所述的移植方法涉及将酶分离的乳腺腺体（乳腺导管上皮和其他能够变形的细胞类型的碎片）或单分散细胞注射到实验鼠2的近亲繁殖、异体或共基因菌株的间状脂肪垫中。由于体间脂肪垫通常没有乳腺组织，它为多个移植部位提供了合适的环境，而无需预先清除内源性上皮。因此，宿主动物的内源性、腹内性乳腺不受手术操作，发育正常，不能干扰结果解释。此外，完整的乳腺可用于比较，以评估宿主与捐赠者对乳腺上皮发育和肿瘤发生的影响18、25。虽然从单个干细胞中重新填充乳腺可供小鼠使用，但尚未为大鼠开发，主要原因是缺乏为大鼠乳腺干细胞选择抗体25、26、27。尽管如此，单分散的乳腺上皮细胞的移植可以成功地进行，并且这些细胞将正常发育时移植到适当的框架2，3，4。虽然有机物适合多种用途，但定量应用需要单分散细胞，例如，确定电离放射治疗后癌症启动所需的乳腺上皮细胞数量，或比较流细胞选择乳腺上皮细胞群的特性29。
迄今为止，这里描述的手术是大鼠进行乳腺移植的最强有力的方法，其总体目标是研究乳腺发育和乳腺癌发展背后的机制。通常，供体和/或受体动物在上皮移植之前、期间或之后会接触到不同的变量。例子包括涉及化学致癌物30的单基因研究，辐射28，31，32，宿主/供体基因组18的基因操作，和荷尔蒙操纵12。本协议中描述的酶分离的一个主要优点是有机会分离上皮有机体或单分散细胞，进行包括流式细胞学、三维培养、基因编辑等的补充实验。该技术的未来应用将包括对供体和/或宿主组织进行遗传工程的额外操作。例如，供体细胞可以使用CRISPR-Cas9基因编辑系统在任何选定的基因组位点进行基因改造。同样，接受者大鼠也可以进行基因改造，以研究供体和受体工程遗传因素之间的相互作用。
Protocol
所有动物都被安置在AAALAC批准的设施中，该协议中描述的实验得到了MUSC机构动物护理与使用委员会（IACUC）的批准。用于互惠移植的动物应为近亲繁殖或异种菌株，具有优选或背交至少6代优异的优生状态。 1. 收获供体大鼠乳腺上皮 确定移植所需的供体大鼠数量。注：一般来说，1只供体大鼠（4周龄）可以提供足够的细胞，用于移植到4个受体动物中。该协议的某些?…
Representative Results
捐赠者和受助人乳腺图1A显示了分离和制备大鼠乳腺上皮细胞进行移植的步骤。在4周大时，供体大鼠的内源性乳腺已经开始成熟，上皮可以形象化在全安装的幻灯片上，上面沾满了明珠（图1B）。在这个年龄，一只供体大鼠将提供大约1 x 106个细胞进行移植?…
Discussion
该协议描述了一种针对大鼠工作而优化的乳腺上皮细胞移植技术。从供体大鼠（3-5周年龄）分离的乳腺上皮器官被移植到受体大鼠的间膜白脂肪垫（也是3-5周的年龄）。结果可以解释为4-6周后，使用光显微镜检查移植组织;然而，在进行完整实验之前，必须确定移植和牺牲之间的最佳时间。如果时间过得太少或过长，结果既不是可解释的，也不是有意义的。为了优化协议，分析移植后6-8周一小组动…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作由霍林斯癌症中心癌症中心支持赠款P30 CA138313试点研究资金资助，来自国家卫生研究院（https://www.nih.gov/），以及南卡罗来纳医科大学病理学和实验室医学系的资助。我们要感谢玛丽妮·史密斯录制采访声明。
Materials
| 0.2 µM syringe filters | Fisher Scientific | 09-715G | sterile-filtering collagenase digestion media |
| 1.5 – 2.0 mL microcentrifuge tubes (sterile) | Fisher Scientific | 05-408-129 | containing resuspended cells and/or brain homogenate mixture |
| 100 µM cell strainers | Corning | 431752 | filtering brain homogenate |
| 100 uL gastight syringes with 25 gauge needles | Hamilton | 81001 & 90525 | For injecting graft mixture into recipient animals (1 per donor genotype/condition) |
| 1000 uL pipette tips + pipette | – | – | transferring cells/mixtures/tissue |
| 15 mL polypropylene tube | Falcon (Corning) | 352196 | brain homogenate mixture storage, or cell : homogenate mixture for transplantation |
| 40 µM cell strainers | Corning | 431750 | filtering organoids after washing the cell pellet |
| 50 mL polypropylene tubes | Fisher Scientific | 05-539-6 | for collagenase digestion of donor mammary gland tissue |
| 60 mm dishes | Thermo Scientific | 130181 | for mincing tissue |
| Alum Potassium Sulfate | Sigma-Aldrich | 243361/237086 | staining mammary gland whole mount slides |
| Anesthesia vaporizer for veterinary use | – | – | follow institutional protocol |
| Beta-dine or iodine | – | – | |
| Borosilicate glass culture tube for homogenization | Fisher Scientific | 14-961-26 | for homogenization of brain (use appropriate tube for homogenizer) |
| Carmine | Sigma-Aldrich | C6152/1022 | staining mammary gland whole mount slides |
| Cell counting apparatus | – | – | |
| Clean animal cages for recovery | – | – | follow institutional protocol |
| Collagenase Type 3 | Worthington Biochemical Corp. | LS004183 | enzymatic digestion of minced mammary gland tissue from donor rats |
| deionized water | – | – | for chemical solutions |
| DMEM/F12 | GIBCO | 11320033 | for mincing tissue, collagenase digestion media and resuspending epithelial cell mixtures |
| EDTA | – | – | monodispersion mixture |
| Ethanol, 200 Proof | Decon Labs | 2705/2701 | mammary whole mount slide fixative, mammary whole mount slide washes, cleaning surgical incision sites (diluted) |
| Fetal Bovine Serum (FBS) | Hyclone | – | inactivation solution |
| Gauze | – | – | |
| Glacial acetic acid | Fisher Scientific | A38-212 | use for mammary whole mount slide fixative (1:4 glacial acetic acid in 100% ethanol) |
| HBSS | GIBCO | – | monodispersion mixture |
| Heating pads | – | – | follow institutional protocol |
| Ice buckets (x2) | – | – | |
| Incubator with orbital rotation | – | – | must be capable of maintaining 37°C, shaking at 220-225 RPM (for collagenase digestion of mammary tissue) |
| Isoflurane anesthesia | – | – | follow institutional protocol |
| Light microscope or digital camera | – | – | visualizing whole mounted mammary epithelium and/or acquiring images |
| Mechanical homogenizer | Fisher Scientific | – | TissueMiser or alternative models |
| Mineral oil, pure | Sigma-Aldrich/ ACROS Organics | 8042-47-5 | long-term storage of cleared mammary gland whole mounts |
| Oxygen tanks for anesthesia vaporizer | – | – | follow institutional protocol |
| Paper towels or delicate task wipes | – | – | |
| Positively-charged microscope slides | Thermo Scientific | P4981-001 | mammary gland tissue whole mounts |
| Postoperative analgesic | – | – | Institutional protocol |
| Scale | body weight measurements of animals, proper dosing of pain medication | ||
| Shaver | – | – | electric clippers, or other |
| Staining jars | – | – | minimum of 1 per chemical wash, size appropriate for the number of slides, glass preferred |
| Sterile field drapes | IMCO | 4410-IMC | used during transplantation |
| Sterile scissors and forceps x3 (autoclaved) | – | – | autoclave surgical tools used for donors and recipients |
| Syringes: 5 mL (or greater) | – | – | for sterile filtration of collagenase digestion media |
| Trypsin | Worthington | monodispersion mixture | |
| Waste collection receptacle for liquids (poured or aspirated) | – | – | |
| Wound clip applier, clips, and removal tool | Fine Science Tools | 12020-00 | Closing the skin incision over the interscapular white pad pad |
| Xylenes | Fisher Scientific | X3S-4 | clearing mammary gland whole mount slides after staining |
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Citer Cet Article
Shunkwiler, L. B., Haag, J. D., Gould, M. N., Smits, B. M. G. Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad. J. Vis. Exp. (157), e60401, doi:10.3791/60401 (2020).