使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞
Summary
该协议的目标是通过使用胶原酶对组织进行酶消化，分离位于结肠层状的单核细胞。该协议允许对单核细胞进行有效分离，从而产生单个细胞悬浮液，进而可用于强健的免疫分体。
Abstract
肠道是体内免疫细胞数量最多的家庭。小型和大型肠道免疫系统可调节外源抗原，并调节对强效微生物衍生的免疫刺激的反应。因此，肠道是许多疾病中免疫调节和炎症的主要靶点，包括但不限于炎症性肠病，如克罗恩病和溃疡性结肠炎、骨后移植物与宿主疾病 （GVHD）骨髓移植（BMT），和许多过敏和传染性疾病。胃肠道炎症和结肠炎的Murine模型被大量用于研究胃肠道并发症和临床前优化预防和治疗策略。通过对肠道免疫细胞的分离和型板分析从这些模型中收集的数据对于进一步了解可应用于改善胃肠道和全身炎症疾病的免疫理解至关重要。本报告描述了使用混合硅基密度梯度界面将单核细胞（MNC）从结肠分离的高效方案。该方法可重复分离大量可行的白细胞，同时最大限度地减少污染碎片，从而允许随后通过流式细胞仪或其他方法进行免疫型型。
Introduction
虽然胃肠道 （GI） 主要致力于处理和重新吸收来自食物的营养物质，但胃肠道也保持在血管、淋巴和神经系统以及许多其他器官的完整性中的核心作用。其粘膜和亚粘膜免疫系统1。GI免疫系统在胃肠道和全身健康中都具有影响作用，因为它经常接触来自食物、共体细菌或入侵病原体1、2的外来抗原。因此，GI免疫系统必须保持一个微妙的平衡，在其中它容忍非致病性抗原，同时适当响应致病性抗原1，2。当耐受性和防御的平衡被打乱时，局部或全身免疫失调和炎症可能导致无数的疾病1，2，3。
肠中至少有70%的淋巴细胞在体内4。大多数初级免疫学相互作用涉及肠道中三个免疫站中的至少一个：1）佩耶尔的贴片，2）皮内淋巴细胞（IEL）和3）拉米纳表体淋巴细胞（LPL）。其中每一个都由一个复杂的免疫细胞互联网络组成，对肠道5的正常免疫挑战作出快速反应。限于肌肉粘膜上方的频闪，松散的层状膜是肠道粘膜的结缔组织，包括软体、血管、淋巴排水和粘膜神经系统的脚手架，以及许多与生俱来和自适应免疫子集6，7，8，9。LPL由CD4+和CD8+T细胞组成，近似比例为2：1，血浆细胞和骨髓体细胞包括，树突状细胞、乳腺细胞、嗜酸性细胞和巨噬细胞6。
人们越来越有兴趣了解肠道的免疫调节和炎症，因为它涉及到各种疾病状态。克罗恩病和溃疡性结肠炎等疾病都表现出不同程度的结肠炎10，11，12。此外，接受异体骨髓移植（Allo-BMT）的骨髓或免疫系统的恶性或非恶性疾病患者可以发展各种形式的结肠炎，包括1）从调理方案直接毒性在BMT之前，2）由BMT和3）移植-宿主疾病（GVHD）后由供体型T细胞驱动，在BMT13、14、15之后对组织中的供体异抗原作出反应，由免疫抑制引起的感染。所有这些后BMT并发症导致肠道的免疫环境显著改变16，17，18。该方法允许对小鼠结肠中的免疫细胞积累进行可靠的评估，在BMT之后应用于小鼠受体时，有助于对参与移植耐受性中的供体和受体免疫细胞进行有效检测19 ，20.肠道炎症的其他原因包括恶性肿瘤、食物过敏或肠道微生物群的中断。该协议允许从结肠进入肠道单核细胞，并经修改后，在临床前小鼠模型中访问小肠的白细胞。
使用搜索词”肠道和免疫细胞和分离”的PubMed搜索揭示了200多个出版物，描述了小肠消化提取免疫细胞的方法。然而，对结肠的类似文献搜索没有产生指定免疫细胞与结肠分离的精心划定的协议。这可能是因为结肠有更肌肉和间质层，使其更难完全消化比小肠。与现有协议不同，该协议专门使用不含其他细菌胶原酶（胶原酶 D/胶原酶 I）的胶原酶 E。我们证明，使用该协议，可以在保持分离的肠道单核免疫细胞（MNC）质量的同时，实现结肠组织的消化，而无需添加抗结块试剂，如醋酸钠（EDTA）、Dispase II和脱氧核糖核酸I（脱氧核糖核酸I）21，22，23 。该协议经过优化，允许从鼠结肠中可重复的强健提取可存活的MNC，以便进一步定向研究，并应有助于研究结肠的免疫学或（有修改）小肠24，25.
Protocol
Representative Results
Discussion
此视觉协议描述了用于分离结肠单核细胞（包括拉米那体淋巴细胞 （LPL）的耐受性好的方法。鉴于此方案在评估严重的移植后小鼠结肠炎模型时进行了优化，其中炎症细胞因子和组织损伤导致恢复的MNC生存能力较差，我们预计这些方法可以转化为其他需要结肠MNC的型态分析的应用程序。这些包括，但不限于评估炎症性肠病小鼠模型中的结肠炎症，研究结肠炎靶向治疗的免疫反应，以及传染性病原…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了#1K08HL088260和#1R01HL133462-01A1（NHLBI）（A.B.P.，H.N.，S.J.）和巴切洛尔儿科研究基金会（D.M.，H.N.，S.J.，A.A.H.，A.B.P.）的支持。本研究中使用的C57BL/6和BALB/c小鼠要么在我们的工厂中繁殖，要么由杰克逊实验室或塔科尼奇提供。
Materials
| 60 mm Petri DIsh | Thermo Scientific | 150288 | |
| 1x PBS | Corning | 21-040-CV | |
| 10x PBS | Lonza BioWhittaker | BW17-517Q | |
| 10 mL Disposable Serological Pipette | Corning | 4100 | |
| 10mL Syringe | Becton Dickinson | 302995 | |
| 15mL Non-Sterile Conical Tubes | TruLine | TR2002 | |
| 18- gauge Blunt Needle | Becton Dickinson | 305180 | |
| 25 mL Disposable Serological Pipette | Corning | 4250 | |
| 40 micrometer pore size Cell Strainer | Corning | 352340 | |
| 50 mL Falcon Tube | Corning | 21008-951 | |
| Bovine Serum Albumin (BSA) | Sigma | A4503-1KG | |
| Fixation Buffer | Biolegend | 420801 | |
| E. coli Collagenase E from Clostridium histolyticum | Sigma | C2139 | |
| EDTA, 0.5M Sterile Solution | Amresco | E177-500ML | |
| Fetal Bovine Serum | Thermo /Fisher Scientific -HyCLone | SV30014.03 | |
| HEPES | GE Healthcare-HyClone | SH30237.01 | |
| Percoll | GE Healthcare-Life Sciences | 1708901 | |
| RPMI Medium | Corning | 17-105-CV | |
| Sodium Azide | VWR Life Science Amresco | 97064-646 | |
| Trypan Blue | Lonza BioWhittaker | 17-942E |
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