胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型
Summary
本方案描述了胫骨肉瘤细胞注射生成具有原位骨肉瘤和肺转移病变的小鼠模型。
Abstract
骨肉瘤是儿童和青少年中最常见的原发性骨癌，肺是最常见的转移部位。骨肉瘤伴肺转移患者的五年生存率低于30%。因此，利用小鼠模型模拟人类骨肉瘤的发展，对于了解骨肉瘤癌发生和肺转移的基本机制，开发新的治疗方法具有重要意义。在这里，报告了 通过胫骨肉瘤细胞的胫内注射产生原发性骨肉瘤和肺转移小鼠模型的详细程序。结合生物发光或X射线实时成像系统，这些活小鼠模型用于监测和量化骨肉瘤的生长和转移。为了建立该模型，将含有骨肉瘤细胞的基底膜基质装入微量注射器中，并在麻醉后注射到每只无胸腺小鼠的一个胫骨中。当原发性骨肉瘤达到IACUC批准的方案中的大小限制时，处死小鼠。将有骨肉瘤的腿部与有转移病变的肺部分开。这些模型的特征是潜伏期短、生长迅速、病变严重以及监测原发性和肺转移性病变发展的敏感性。因此，这些是探索骨肉瘤癌发生和肺转移中特定因素的功能和机制，肿瘤微环境，评估 体内治疗效果的理想模型。
Introduction
骨肉瘤是儿童和青少年中最常见的原发性骨癌1，2，其主要浸润于周围组织，甚至在诊断患者时转移至肺部。肺转移是骨肉瘤治疗的主要挑战，骨肉瘤肺转移患者的五年生存率仍低至20%-30%3，4，5。然而，自 20 世纪 70 年代以来，由于引入了化疗 6，原发性骨肉瘤的五年生存率已提高到约 70%。因此，迫切需要了解骨肉瘤癌癌和肺转移的基本机制，以开发新的疗法。最能模拟人类骨肉瘤进展的小鼠模型的应用具有重要意义7.
骨肉瘤动物模型是通过自发的，诱导的基因工程，移植和其他技术产生的。自发性骨肉瘤模型由于肿瘤形成时间长、肿瘤发生率不一致、发病率低、稳定性差等原因，很少使用8，9。虽然诱导骨肉瘤模型比自发性骨肉瘤更容易获得，但诱导骨肉瘤模型的应用是有限的，因为诱导因子会影响骨肉瘤10的微环境、发病机制和病理特征。转基因模型有助于了解癌症的发病机制，因为它们可以更好地模拟人体生理和病理环境;然而，转基因动物模型也由于转基因修饰的难度大，长期且成本高而存在局限性。而且，即使在最广泛接受的由p53和Rb基因修饰产生的转基因动物模型中，也只有13.6%的肉瘤发生在四肢骨11，12中。
移植是近年来最常用的原发性和远处转移性癌症模型生成方法之一，因为它具有操作简单，肿瘤形成速率稳定，均匀性较好13。移植包括异位移植和根据移植部位的原位移植。在骨肉瘤异位移植中，骨肉瘤细胞被注射到动物的原发性骨肉瘤部位（骨）之外，通常在皮下，皮下14。虽然异位移植很简单，不需要在动物身上进行手术，但注射骨肉瘤细胞的部位并不代表实际的人类骨肉瘤微环境。骨肉瘤原位移植是将骨肉瘤细胞注射到动物的骨骼中，如胫骨15，16。与异位移植物相比，原位骨肉瘤移植物的特点是潜伏期短，生长快，侵蚀性强;因此，它们是骨肉瘤相关研究的理想动物模型17。
最常用的动物是老鼠，狗和斑马鱼18，19。骨肉瘤的自发模型通常用于犬类，因为骨肉瘤是犬科动物中最常见的肿瘤之一。但是，由于肿瘤形成时间长，肿瘤发生率低，均匀性差，稳定性差，该模型的应用受到限制。斑马鱼通常用于构建转基因或敲除肿瘤模型，因为它们的繁殖速度很快20。但斑马鱼基因与人类基因不同，因此其应用有限。
这项工作描述了 通过在 胸腺小鼠中通过胫骨内注射骨肉瘤细胞在胫骨中产生肺转移的原发性骨肉瘤的详细程序，注意事项和代表性图像。该方法应用于小鼠胫骨原发性骨肉瘤进行治疗效果评价，其重现率高达21，22。
Protocol
所有动物实验均经上海中医药大学动物福利委员会批准。四周龄的雄性BALB / c无胸腺小鼠在手术前适应一周，以进行骨肉瘤细胞的原位注射。将小鼠饲养在单独通风的小鼠笼中，每个笼子有五只小鼠，在12小时的光照/黑暗循环中，随意获得SPF饲料和无菌水。 1. 细胞的制备 在骨肉瘤细胞（143B-荧光素酶）注射当天，用PBS（pH 7.4）洗涤在10cm细胞培养皿中培养?…
Representative Results
成功的原位（原发性）骨肉瘤和转移性肺模型取决于骨肉瘤细胞的准确原位注射。在这里， 通过 胫骨内骨肉瘤细胞注射成功开发了一种原位（原发性）骨肉瘤模型。 图3A 显示了具有代表性的小鼠携带原位（原发性）骨肉瘤， 图3B 显示了具有代表性的孤立性原位（原发性）骨肉瘤。用卡尺每周测量一次肿瘤体积，并按照步骤2.11中所述计算（<stro…
Discussion
原位注射骨肉瘤细胞是研究骨肉瘤癌发生中特定因素的功能和机制以及开发以评估治疗效果的理想模型。为避免肿瘤生长的差异，将大多数与相同数量汇合的80%-90%的活性骨肉瘤细胞小心地注射到每只小鼠的胫骨中，严格控制细胞胰蛋白酶消化时间而不影响细胞活力。由于细胞团块会影响细胞计数，导致将不准确的细胞数量注射到每只小鼠的胫骨中，因此需要用移液管适当地上下混合细胞悬浮液，…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究由（1）中国国家重点研发计划（2018YFC1704300和2020YFE0201600），（2）国家自然科学基金（81973877和82174408）资助。
Materials
| Automatic cell counter | Shanghai Simo Biological Technology Co., Ltd | IC1000 | Counting cells |
| Anesthesia machine | Shenzhen RWD Life Technology Co., Ltd | R500IP | The Equipment of Anesthesia mice |
| BALB/c athymic mice | Shanghai SLAC Laboratory Animal Co, Ltd. | / | animal |
| Basement Membrane Matrix | Shanghai Uning Bioscience Technology Co., Ltd | 356234, BD, Matrigel | re-suspende cells |
| Bioluminescence imaging system | Shanghai Baitai Technology Co., Ltd | Vieworks | tracking the tumor growth and pulmonary metastasis, if the injection cell is labeled by luciferase |
| Centrifuge tube (15 mL) | Shanghai YueNian Biotechnology Co., Ltd | 430790, Corning | Centrifuge the cells |
| isoflurane | Shenzhen RWD Life Technology Co., Ltd | VETEASY | Anesthesia mice |
| MEM media | Shanghai YueNian Biotechnology Co., Ltd | LM-E1141 | Cell culture medium |
| Micro-volume syringe | Shanghai high pigeon industry and trade Co., Ltd | 0-50 μL | Inject precise cells into the tibia |
| Phosphate-buffered saline | Beyotime Biotechnology | ST447 | wash the human osteosarcoma cells |
| 1ml syringes | Shandong Weigao Group Medical Polymer Co., Ltd | 20200411 | drilling |
| 143B cell line | ATCC | CRL-8303 | osteosarcoma cell line |
| Trypsin (0.25%) | Shanghai YueNian Biotechnology Co., Ltd | 25200056, Gibco | trypsin treatment of cells |
| Trypan blue | Beyotime Biotechnology | ST798 | Staining cells to assess activity |
| vector (pLV-luciferase) | Shanghai YueNian Biotechnology Co., Ltd | VL3613 | Plasmid |
| Lipofectamine 2000 | Shanghai YueNian Biotechnology Co., Ltd | 11668027,Thermo fisher | Plasmid transfection reagent |
| X-ray imaging system | Brook (Beijing) Technology Co., Ltd | FX PRO | X-ray images were obtained to detect tumor growth |
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Citer Cet Article
Chang, J., Zhao, F., Sun, X., Ma, X., Zhi, W., Yang, Y. Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models. J. Vis. Exp. (176), e63072, doi:10.3791/63072 (2021).