成年小鼠黏膜下层和固有层肠道胶质细胞的分离
Summary
在这里, 我们描述了肠道黏膜下层的肠神经胶质细胞的分离, 使用顺序 EDTA 孵化螯合价阳离子, 然后孵化在非酶细胞恢复解决方案。在聚 d-赖氨酸和层粘连蛋白的作用下, 电镀细胞悬浮物会导致黏膜下胶质细胞的高度富集培养, 用于功能分析。
Abstract
肠道神经系统 (电梯) 由位于平滑肌壁、黏膜下层和固有层内的神经细胞和肠胶质细胞组成。电梯在肠道动态平衡中发挥重要作用, 通过释放各种营养因素, 促进上皮屏障的完整性。大多数对原发性肠神经胶质细胞的研究都是在酶解离肌间丛丛中分离出来的。本文介绍了一种非酶法分离和培养电梯肠黏膜下层和固有层的方法。在人工清除纵向肌肉层后, 电梯从固有膜和黏膜下层解放出来, 使用顺序 HEPES 缓冲 EDTA 孵化, 然后在商业上可用的非酶细胞回收解决方案孵化。EDTA 孵化足够从固有层剥离大部分上皮粘膜, 使细胞恢复解决方案解放黏膜下电梯。任何残余的固有层和平滑的肌肉被丢弃连同肌间丛胶质细胞。电梯很容易通过表达胶质纤维酸性蛋白 (GFAP) 的能力来鉴别。只有约50% 的细胞悬浮液中含有 GFAP + 细胞完成组织孵化后, 在电镀之前, 聚 d-赖氨酸/层粘连蛋白基底。然而, 经过3天的培养细胞的神经胶质细胞源性神经营养因子 (GDNF) 包含培养基, 细胞的数量附着在基板涂层板组成的 > 95% 肠道胶质。我们创建了一个混合的鼠标线, 通过培育 hGFAP 鼠到罗莎-tdTomato 的记者线, 以跟踪的百分比的 GFAP + 细胞使用内源性细胞荧光。因此, 非肌间丛肠胶质细胞可通过非酶法分离, 培养至少5天。
Introduction
由于其在肠道完整性和稳态1,2中公认的作用, 对肠道胶质细胞 (电梯) 功能的兴趣稳步增加。另外, 电梯根据他们的位置沿胃肠道3,4的长度变化。电梯释放各种营养因子, 包括胶质细胞源性神经营养因子 (GDNF), 有助于肠道动力1,5 , 并对微生物副产物6,7作出反应。研究表明, 胃癌的种群是异构的, 它们的功能取决于它们是黏膜下的还是驻留在肌间丛丛1,7。例如, 黏膜下层的电梯有助于紧密连接8。电梯中的差异性 GFAP 表达和磷酸化与帕金森氏病有关, 表明它们可能与这种疾病的肠道表型有联系9。最近, 据观察到, 从近端肠道黏膜下层电梯分离培养的核蛋白叶普斯镇的丢失足以诱发激素胃泌素10的表达。因此, 有人建议电梯可能是十二指肠 gastrinomas 的起源, 一种神经内分泌肿瘤10。总的来说, 这些例子突显了研究孤立电梯在神经病理性疾病和癌症11中的行为和功能的相关性。
这一领域的挑战仍然是如何在体外分离和研究胃癌种群。血统痕迹实验表明, 电梯在黏膜下层和固有层的起源于祖细胞在肌间丛丛7。尽管有几种已发布的隔离协议可用于生成肌间丛电梯12、13、14、15、16、17的区域性, 18,19, 没有明确的目标是孤立的黏膜下/叶片固有胃癌人口。现有的胃癌隔离协议专门使用机械分离或平滑肌肉的显微解剖结合酶解分离, 最终丢弃粘膜细胞层。
本手稿的目的是为了证明从固有层的非酶分离初级电梯的步骤,在体外研究。由于没有明确区分肌间丛电梯与黏膜下层的标记, 利用平滑肌上皮黏膜的空间分离来分离黏膜下电梯。此外, 通过将 EDTA 螯合剂与非酶解分离相结合, 电梯从黏膜下层分离出来, 与平滑肌相比较, 与肌间丛电梯。在神经胶质细胞基质上培养细胞,如聚 d-赖氨酸和层粘连蛋白, 进一步分离黏膜下层和固有层电梯。
Protocol
Representative Results
Discussion
电梯在肠道动态平衡中起着重要作用,在体外分离和研究是十分必要的。在该协议中, 介绍了一种从成年小鼠肠道固有层中分离电梯的简便方法, 研究肠神经胶质功能。
用棉签去除粘附的肠系膜和 LMMP, 可以去除位于纵、圆肌之间的肌间丛胶质细胞, 增加缓冲液对黏膜下表面的可接触性, 并去除大部分大毛细血管.后者减少了红细胞的数量, 污染了最终的文化。本系列 EDTA 孵?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者希望承认来自 R37 DK045729 (JLM)、R01 AR060837 (to) 和密歇根大学胃肠研究中心分子核心 P30 DK034933 的支持。
Materials
| Poly-D lysine (1 mg/ml stock) | Sigma | A-003-E | Dilute 1:10 |
| Laminin (0.5 mg/ml stock) | Sigma | L4544 | Dilute to 10 µg/mL on ICE |
| EDTA (0.5M) | Lonza | 51201 | Dilute 1:100 in DPBS |
| HEPES (1 M) | Corning | 36216004 | Dilute 1:100 in DPBS |
| Cell Recovery Solution | Corning | 354253 | |
| Dulbecco's Phosphate Buffered Saline (DPBS) | HyClone | SH30028.02 | |
| DMEM/F-12 | Thermo Fisher Scientific | 11320033 | |
| Penicillin-Streptomycin (100X) | Life Technologies | 15140-122 | |
| Gentamicin (50mg/mL stock) | Life Technologies | 15750060 | |
| GDNF (10 µg stock) | Sigma | SRP3200 | |
| L-Glutamine (200 mM stock) | Life Technologies | 25030-081 | |
| Chicken anti-GFAP | Thermo Fisher Scientific | PA1-10004 | |
| Goat anti-a-Smooth Muscle Actin | Abcam | ab112022 | |
| Mouse anti-Pgp9.5 | Novus Biologicals | NB600-1160 | |
| Goat anti-E-cadherin | R&D Systems | AF748 | |
| Rabbit S100 | Abcam | ab34686 | |
| Mouse p75 NTR | Millipore | MAB5592 | |
| Alexa Fluor 488 Goat Anti-Chicken IgY | Invitrogen | A-11039 | |
| Alexa Fluor 488 Donkey Anti-Goat IgG | Invitrogen | A-11055 | |
| Alexa Fluor 568 Goat Anti-Mouse IgG | Invitrogen | A-11004 | |
| Alexa Fluor 594 Donkey Anti-Rabbit IgG | Invitrogen | R-37119 | |
| Prolong Gold antifade Reagent with DAPI | Thermo Fisher Scientific | P36931 | |
| Fungizone (Amphotericin B) 250 µg/ml | Life Technologies | 15290-018 | |
| L-Fura-2-AM | Invitrogen | F-14201 | |
| CCK peptide | Anaspec, Fremont, CA | AS-20741 | |
| Gastrin peptide (Gastrin-17) | Abbiotec, Bloomington, IN | 350188 | |
| Nylon Mesh Celll Strainer (100 µm) | Fisher Scientific | 22363549 | |
| Nylon Mesh Celll Strainer (40 µm) | Fisher Scientific | 22363547 | |
| Disposable Serologic Pipet 5 ml | Fisher Scientific | 13-678-11D | |
| 0.25% Trypsin-EDTA (1X) | Life Technologies | 25200-056 |
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