原代小鼠骨骼肌微血管内皮细胞的分离
Summary
骨骼肌微血管内皮细胞 (mmec) 塑造了肌肉毛细血管的内壁, 并调节两者, 流体分子的交换和 (免疫) 细胞在肌肉组织和血液之间的迁移。分离原生小鼠 mmeec, 如下所述, 使 “我的血管单位” 能够进行全面的体外调查。
Abstract
骨骼肌毛细血管内皮细胞 (肌肉微血管内皮细胞, mmec) 在调节液体和营养物质的交流以及对感染的免疫反应之间建立了障碍通过控制免疫细胞迁移的代理。对于这些功能, mmec 形成一个功能性的 “肌血管单位” (mvu), 具有更多的细胞类型, 如成纤维细胞、周细胞和骨骼肌细胞。因此, mmec 的功能障碍, 因此 mvu 有助于各种各样的肌病。然而, mmec 在健康和疾病方面的监管机制仍然没有得到充分的了解, 它们的阐明先于治疗肌病的更具体的方法。在 mvu 的背景下对主要 mmec 功能进行隔离和深入调查, 可能有助于更好地了解这些过程。
本文提供了一种通过机械和酶解分离分离骨骼肌原生小鼠 mmec 的方案, 包括纯化和培养维持步骤。
Introduction
通过血液, 细胞和器官提供氧气、底物和其他必要的分子。这种交换发生在毛细血管中, 是最小的船只。毛细血管是由内皮细胞 (ec) 内层形成的, 其完整性仍然是成功调节血管内和间质间隙之间肌肉稳态的先决条件。为了确保可溶性因子和细胞的选择性过渡, ec 构成了一个单层连接的紧密和粘附连接 1. 除了作为营养或代谢产物的屏障的作用外, ec 还调节炎症过程中白细胞的吸收。炎症或组织损伤导致 ec 表面粘附分子的上调, 并产生促进白细胞附着和转移到目标组织2的趋化因子.因此, ec 严格参与炎症过程的调节, 如防御病原体或组织修复。
ec 功能障碍与血管疾病、慢性肾功能衰竭、静脉血栓形成严重的病原体感染直接相关。此外, ec 几乎总是参与器官特异性自身免疫, 如糖尿病或多发性硬化症3。因此, 血液和器官之间的屏障功能是由不同细胞类型的协同相互作用控制的。在骨骼肌微血管内皮细胞 (mmec) 与肌肉细胞、成纤维细胞和周细胞形成一个功能单元, 即 “myovascular 单位” (mvu)。因此, mvu 功能障碍可能在肌病的病理生理学中起着至关重要的作用。然而, 对这些监管机制的更深入了解仍然缺失, 目前无法确定肌病中新的、急需的治疗目标。
为了研究复杂的生理和病理生理机制, 动物模型是常用的。然而,体外模型通过排除各种混淆因素, 提供了关注感兴趣的主题的优势。为了研究体外过程, 有必要分离出纯和有活力的原代细胞。与细胞系不同的是, 从转基因动物身上分离出的原代细胞能够研究体外基因修饰的后果。
本文介绍了一种利用机械和酶解分离的方法, 然后采用磁性活化细胞分选技术 (mcs) 进行纯化。为此, 使用了针对特定表面标记的磁珠。血小板内皮细胞粘附分子-1 (pecam1, cd31) 主要在 ec 上表达, 可用于丰富这种细胞类型。为了保证高细胞纯度, 对蛋白酪氨酸磷酸酶受体 c 型 (ptprc, cd45) 的否定选择排除了造血源细胞。此外, 还介绍了质量控制、原生小鼠 mmec 的培养、潜在的应用和局限性以及特殊注意事项。
Protocol
Representative Results
Discussion
微血管内皮细胞在所有组织中提供屏障功能, 其功能障碍导致相关器官的疾病3。此外, 针对器官的微血管 ec 研究可以为新的治疗策略铺平道路。因此, 深入了解生理和病理生理条件下的微血管 ec 功能具有重要的科学意义。白细胞与内皮细胞相互作用的调节成功地用于治疗多发性硬化症患者与纳珠单抗, 一种抗体, 防止淋巴细胞粘附, 从而转染 6. 然而, 包括炎症亚?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 “else kröner-fre舍enius-stibtung” (2018_A03 到 tr) 的支持, “创新 medizinische forschung (基金组织) münster” (i-ru2001 1811 to tr) 和德国研究基金会 (dfg, inst 2105/27-1–, me 328—1, me 3281-1-1 至 sgm)。黑克·布鲁姆提供的插图图片。
Materials
| 0,25% Trypsin-EDTA | Thermo Fisher | 25200-056 | ready to use |
| ACK buffer | 150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA in water at a pH of 7.3 | ||
| Anti-mouse CD31-FITC (clone MEC13.3) | Biolegend | 102506 | Isotype control: FITC Rat IgG2a, κ Isotype Ctrl |
| Anti-mouse CD45-PE (clone 30-F11) | Biolegend | 103106 | Isotype control: PE Rat IgG2b, κ Isotype Ctrl |
| bFGF | Peprotech | 100-18B | Basic fibroblast growth factor |
| BSA | Sigma Aldrich | A4503 | |
| CD31 MicroBeads mouse | Miltenyi Biotec | 130-097-418 | |
| CD45 MicroBeads mouse | Miltenyi Biotec | 130-052-301 | |
| Collagenase-Dispase | Roche | 10269638001 | Collagenase from V. alginolyticus, Dispase from B. polymyxa |
| Corning Costar TC-Treated Multiple 6-Well Plates | Corning | 3516 | |
| Cy3-conjugated anti-rat IgG antibody | dianova | 712-166-153 | |
| DAPI (ProLong Gold antifade reagent with DAPI) | Thermo Fisher | P36935 | |
| Desoxyribonuclease | Sigma Aldrich | D4513 | Deoxyribonuclease I from bovine pancreas |
| Diethylpyrocarbonat treated water | Thermo Fisher | AM9916 | |
| DMEM, containing Glutamin Supplement and pyruvate | Thermo Fisher | 31966-021 | warm up to 37 °C before use |
| dNTP Mix (10 mM) | Thermo Fisher | R0192 | 1 mL |
| EDTA | Sigma Aldrich | E5134 | |
| FACS tubes | Sarstedt | 551,579 | |
| Falcon 70 μm Cell Strainer | Corning | 352350 | |
| FC buffer | 0.1% BSA, 0.2% NaN3, 2 mM EDTA | ||
| Fetal calf serum | Sigma Aldrich | F6178 | Fetal calf serum |
| Fixable Viability Dye eFluor780 | Thermo Fisher | 65-0865-14 | |
| Forceps (serrated, straight, 12 cm) | Fine Science Tools | 11002-12 | |
| Forceps (serrated, straight, 12 cm) | Fine Science Tools | 11009-13 | |
| Insulin syringe 100 Solo 1ml (Omnifix) | Braun | 9161708V | |
| large magnetiv columns (LS columns) | Miltenyi Biotec | 130-042-401 | for CD45-MACS-step |
| MCS buffer | 0.5% BSA, 2 mM EDTA in PBS at a pH of 7.2 | ||
| Medium magnetic column (MS column) | Miltenyi Biotec | 130-042-201 | for CD31-MACS-step |
| Nuclease free water | Thermo Fisher | R0581 | |
| PBS | Sigma Aldrich | Phosphate buffered saline, ready to use | |
| PCR buffer (5x) | Thermo Fisher | EP0742 | in a kit with the reverse transcriptase |
| Pecam1 rat α-mouse | SantaCruz | Sc-52713 | 100 µg/mL |
| Penicillin-Streptomycin | Sigma Aldrich | P4333 | |
| primary murine muscle cells | celprogen | 66066-01 | |
| Primer Cdh15 (M-Cadherin) | Thermo Fisher | Mm00483191_m1 | FAM labeled |
| Primer Cldn5 (claudin-5) | Thermo Fisher | Mm00727012_s1 | FAM labeled |
| Primer Ocln (occludin) | Thermo Fisher | Mm00500912_m1 | FAM labeled |
| Primer Pax-7 | Thermo Fisher | Mm01354484_m1 | FAM labeled |
| Primer Tjp-1 (Zonula occludens 1) | Thermo Fisher | Mm00493699_m1 | FAM labeled |
| Primer 18s rRNA (Eukaryotic endogenous control) | Thermo Fisher | 4310893E | VIC labeled |
| qPCR buffer (Maxima Probe/ROX qPCR Master Mix (2X) | Thermo Fisher | K0231 | 2 x 1,25 mL; for 200 reactions each |
| Random mixture of single-stranded primer | Thermo Fisher | SO142 | Random Hexamer Primer |
| Reverse Transcriptase (200 U/μL) + PCR buffer (5x) | Thermo Fisher | EP0742 | |
| Rnase Inhibitor (40 U/μL) | Thermo Fisher | EO0381 | |
| Scissor (cutting edge 23 mm, sharp/sharp) ) | Fine Science Tools | 14088-10 | |
| Scissor (cutting edge 42 mm, sharp/blunt) | Fine Science Tools | 14001-13 | |
| Speed Coating solution | PeloBiotech | PB-LU-000-0002-00 |
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