用于血管生成研究 的改良体内 基质凝胶塞测定
Summary
这里介绍的方法可以在不染色的情况下评估 试剂对体内 血管生成或血管通透性的影响。该方法使用右旋糖酐-FITC通过尾静脉注射来观察新血管或血管渗漏。
Abstract
已经开发了几种模型来研究 体内血管生成。然而，这些模型大多复杂且昂贵，需要专门的设备，或者难以进行后续定量分析。在这里，我们提出了一种改良的基质凝胶塞测定法来评估 体内血管生成。在该方案中，在存在或不存在促血管生成或抗血管生成试剂的情况下，将血管细胞与基质凝胶混合，然后皮下注射到受体小鼠的背部。7天后，通过尾静脉注射含有葡聚糖-FITC的磷酸盐缓冲盐水并在血管中循环30分钟。收集基质凝胶塞并用组织包埋凝胶包埋，然后切割 12 μm 切片用于荧光检测，无需染色。在该测定中，高分子量（~150,000 Da）的葡聚糖-FITC可用于指示功能性血管以检测其长度，而低分子量（~4,400 Da）的葡聚糖-FITC可用于指示新血管的渗透性。总之，该方案可为 体内血管生成的定量研究提供一种可靠、方便的方法。
Introduction
血管生成是从预先存在的血管形成新血管的过程，在许多生理和病理过程中起着至关重要的作用，例如胚胎发育、伤口愈合、动脉粥样硬化、肿瘤发展等1,2,3,4,5。这种动态过程涉及几个步骤，包括基质的降解、血管细胞增殖、迁移和自组织以形成管状结构以及新血管的稳定 6。促进血管生成已被证明在治疗心肌梗塞、中风和其他类型的缺血性疾病中至关重要7，而抑制血管生成被认为是治疗癌症8和类风湿性疾病的有前途的策略9。血管生成被认为是药物发现的组织原则10.因此，构建一种可靠且方便的方法来评估血管生成的程度对于血管生成依赖性疾病的机械研究或药物发现至关重要。
已经开发了几种体外和体内模型来评估血管生成11。其中，二维（2-D）模型，如基质凝胶管形成测定12，不能形成功能性管状结构。动物模型，如后肢缺血模型13,14，可以再现血管生成过程，但很复杂，需要激光散斑血流成像系统。血管形态发生的 3D 模型，如基质凝胶塞测定，提供了一个简单的平台，可以模拟体内血管生成的过程 15，但血管生成的检测需要免疫组化或免疫荧光染色 16,17,18，这些方法可变且可视化不佳。
在这里，我们描述了一种改良的基质凝胶塞测定方案，其中将血管细胞与基质凝胶混合并皮下注射到小鼠背部以形成塞子。在塞子中，血管细胞需要降解基质、增殖、迁移和自组织，最终形成内部环境中有血流的功能性血管。此后，通过尾静脉注射荧光标记的葡聚糖，流经塞子，并将标记可视化以指示新血管。血管生成的内容可以通过血管的长度来定量评估。该方法可以形成在二维血管生成模型12中无法产生的功能性血管，并且不需要像普通基质凝胶塞测定11那样复杂的染色过程。它也不需要昂贵的特定仪器，如后肢缺血模型13、14、19 中的激光散斑血流成像系统。该方法用途广泛、成本低、可量化且易于执行，可用于确定药物的促血管生成或抗血管生成能力，或用于涉及血管生成的机械研究。
Protocol
Representative Results
Discussion
我们提出了一种可靠且方便的方法，用于在不染色 的情况下定量评估体内 血管生成。在该方案中，在促血管生成或抗血管生成试剂存在下将血管细胞与基质凝胶混合，然后皮下注射到Nu / Nu小鼠的背部以形成凝胶塞（图1）。凝胶栓形成7天后，静脉注射葡聚糖-FITC循环30分钟。收集凝胶塞并用组织包埋凝胶包埋，并切片 12 μm 用于摄影（图 2）。葡…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由浙江省自然科学基金（LY22H020005）和中国国家自然科学基金（81873466）资助。
Materials
| Adhesion Microscope Slides | CITOTEST | 188105 | |
| Anesthesia System | RWD | R640-S1 | |
| Cell Counter | Invitrogen | AMQAX1000 | |
| Cell Culture Dish | Corning | 430167 | |
| Cryoslicer | Thermo Fisher | CryoStar NX50 | |
| Dextrans-FITC-150kDa | WEIHUA BIO | WH007N07 | |
| Dextrans-FITC-4kDa | WEIHUA BIO | WH007N0705 | |
| Embedding Cassettes | CITOTEST | 80203-0007 | |
| Endothelial Cell Medium | ScienCell | 35809 | |
| Endothelial Growth Supplements | ScienCell | 1025 | |
| Fetal Bovine Serum | Gibco | 10100147C | |
| Fibroblast Growth Factor 1 | AtaGenix | 9043p-082318-A01 | FGF1 |
| Fluorescence Microscope | Nikon | ECLIPSE Ni | |
| Heating Pad | Boruida | 30-50-30 | |
| Insulin Syringe | BD | 300841 | |
| Isoflurane | RWD | R510-22-10 | |
| Laboratory Balance | Sartorius | BSA124S-CW | |
| Matrigel | Corning | 356234 | Matrix gel |
| Medium 199 powder | Gibco | 31100-035 | |
| Microtubes | Axygen | MCT-150-C | |
| Optimal Cutting Temperature (OCT) Compound | SUKURA | 4583 | Tissue embedding gel |
| Palmitate Acid | KunChuang | KC001 | |
| Penicillin-Streptomycin Liquid | Solarbio | P1400 | |
| Phosphate Buffer Saline | Solarbio | P1022 | |
| Surgical Instruments | RWD | RWD | |
| Tail Vein Injection Instrument | KEW BASIS | KW-XXY | |
| Trypsin-EDTA Solution | Solarbio | T1320 | |
| Ultra-Low Temperature Freezer | eppendorf | U410 | |
| Vascular Endothelial Growth Factor | CHAMOT | CM058-5HP | VEGF |
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