成年小鼠泪液和下颌腺肌上皮细胞的分离
Summary
泪腺 (LG) 有两种细胞类型表达α平滑肌肌动蛋白 (αSMA): 肌上皮细胞 (Mec) 和周周细胞。Mec 是外胚层的起源, 发现在许多腺体组织, 而周围的血管平滑肌细胞的内皮起源。该协议将 Mec 和 pericytes 与小鼠 Lg 隔离开来。
Abstract
泪腺 (LG) 是一种外分泌的管状腺, 分泌一层泪膜水层。LG 上皮树由腺泡、导管上皮细胞和肌上皮细胞 (Mec) 组成。Mec 表达α平滑肌肌动蛋白 (αSMA), 并具有收缩功能。它们存在于多个腺体器官中, 属于外胚层。此外, LG 还含有 SMA + 内皮源血管平滑肌细胞, 称为周长: 包围血管管表面的收缩细胞。一项新的协议允许我们从成年小鼠 Lg 和下颌腺体 (Smg) 中分离出 Mec 和外周。该协议基于Mec 和 pericytes 的基因标记使用 smacret2\+: rosa26-tdtomaatofl/fl 小鼠菌株, 然后制备用于荧光活化细胞分选的 lg 单细胞悬浮液 (facs)).该协议允许根据 Mec 表达的上皮细胞粘附分子 (EpCAM) 分离这两个不同来源的细胞群, 而 pericytes 不表示 EpCAM。分离细胞可用于细胞培养或基因表达分析。
Introduction
肌上皮细胞 (Mec) 存在于许多外分泌腺体中, 包括泪液、唾液、哈德里安、汗液、前列腺和乳房。Mec 是一种独特的细胞类型, 结合了上皮和平滑肌表型。Mec 表达α平滑肌肌动蛋白 (sma), 并具有收缩功能1,2。除 Mec 外, 泪腺 (LG) 和颌下腺 (SMG) 还含有称为外周的 SMA + 血管细胞, 它们是内皮源细胞, 包裹血管管表面3。虽然 mec 和 pericytes 表达了许多标记, 但 sma 是在其他 lg 和 smg 细胞1、3 中唯一没有表达的标记。
在过去40年中, 一些实验室报告了不同外分泌腺组织的离解检测方法, 其中采用了非酶法和酶法。在1980年发表的第一份报告中, 弗里茨和合著者描述了一种使用胶原蛋白/胰蛋白酶溶液中的顺序消化分离猫科动物腮腺的协议.1989年, Hann 和合著者使用胶原酶、透明质酸酶和 DNase5的混合物调整了该协议, 以便从大鼠 lg 中分离出 acini。1990年, 克里普斯和他的同事们公布了泪腺6的非酶解离方法。后来, 在 1998年, Zoukhri 和合著者回到了酶分离协议, 以跟踪 LG 和 SMG 隔离 acini 7 上的 Ca2 +成像.在过去的十年里, 研究人员将注意力转向了从外分泌腺体中分离干细胞祖细胞。普林格尔和合著者在2011年描述了一项分离小鼠 SMG 干细胞的协议。该方法是在分离培养中保持的含干细胞盐虫的基础上进行的。作者声称, 表达干细胞相关标记物的增殖细胞可以从这些盐球8中分离出来.Shatos 和合著者发布了利用酶消化和收集 “释放” 细胞9从未受伤的成年大鼠 Lg 中分离祖细胞的方案.后来, 在 2015年, 阿克曼和合著者调整了这一程序, 以分离推定的 “小鼠泪腺干细胞” (“Mlgsc”), 这些细胞可以作为单层培养在多个通道10上传播。然而, 上述程序都不允许区分细胞亚型和孤立上皮细胞的个体群。2016年, 格罗莫娃和合著者公布了利用流式细胞仪11从成年小鼠 lg 中分离 lg 干细胞的程序。但是, 此协议并不是为了隔离 Mec。
最近, 我们已经证明, 我们能够从3周大的 SMA-GFP 小鼠12只中分离出 SMA+ 细胞。然而, 在这个时候, 我们还没有分离不同的 SBA + 细胞的种群。在这里, 我们建立了一个新的程序, 直接隔离分化的 Mec 和 perictes 从成人 Lg 和 Smg。
Protocol
所有动物工作都是根据国家卫生研究所 (NIH) 的指导方针进行的, 并得到了斯克里普斯研究所动物护理和使用机构委员会的批准。尽一切努力尽量减少老鼠的数量和它们的痛苦。所有实验动物都接受了标准饮食, 可以免费获得自来水。 请注意:图 1A-F示意图概述了 mec 和周二叶隔离的主要步骤。表 1描述了用于此过程的所有试剂和设备。 <p …
Representative Results
鼠标模型隔离 SMA + Mec 和 perices既定协议允许将两个纯种群隔离: Mec 和 perictes 与 Lg 和 Smg (见表 1)。这两种类型的细胞具有不同的大小和外观。微血管周长, 在毛细血管壁周围发展 (图 5a), 并有一个平方状 (图 5A), 而 mec 围绕 lg 分泌阿奇尼, 有很长的过程, 并占据一个相对较大的区域 (图 5…
Discussion
这份手稿描述了一个协议的 MEC 和过氧化物隔离从 LG 和 SMG。该程序是基于 SMA 的遗传标记, 这是唯一可靠的 mec 和果皮生物标志物。
制定这一议定书的紧迫性是由于几乎完全没有文献强调 Mec 与小鼠 Lg 和 Smg 的隔离。虽然以前使用过基因标记, 使用 SMA-GFP 小鼠从3周大的年轻 lg 12 中分离 sma + 细胞, 但由于成年小鼠信号的部分丢失, 不允许使用这些年龄较大的小?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢 Ivo Kalajzic 博士为我们提供了 Smacreert2 小鼠株, Takeshi umazume 为小鼠尾随和基因分型, 马克雪莱获取了图2的专业图片。我们还感谢斯克里普斯科学编辑委员会和马克·雪莱的科学英语编辑。我们感谢斯克里普斯研究所流式细胞仪核心协助细胞分类, 并感谢罗宾·威伦布林博士就流式细胞仪数据分析提供的多次讨论建议。
这项工作得到了国家卫生研究院、国家眼科研究所的支持, 该研究所向 h. p. m. 提供了 5 R01 EY026202 和 1 R01 EY028983 至 h. p. m。
Materials
| Biosafety Cabinet | SterilCard Baker | 19669.1 | Class II type A/B3 |
| 10 ml Disposable serological pipets | VWR | 89130-910 | Manufactured from polystyrene and are supplied sterile and plugged |
| 10 mL Disposable serological pipets | VWR | 89130-908 | Manufactured from polystyrene and are supplied sterile and plugged |
| 15 mL High-clarity polypropylene conical tubes | Falcon | 352196 | |
| 25 mL Disposable serological pipets | VWR | 89130-900 | Manufactured from polystyrene and are supplied sterile and plugged |
| 5 mL FACS round-bottom tubes | Fisher Scientific, Falcon | 14-959-11A | |
| 50 mL High-clarity polypropylene conical tubes | Falcon | 352070 | |
| Antibiotic-antimycotic | Invitrogen | 15240-062 | |
| Appropriate filter and non-filter tips | Any available | Any available | |
| BD Insulin Syringes | Becton Dickinson | 328468 | with BD Ultra-Fine needle ½ mL 8 mm 31G |
| BD Syringes 10 mL | Becton Dickinson | 309604 | Sterile |
| Brilliant Violet 421 anti mouse CD326 (EpCAM) | Biolegend | 118225 | Monoclonal Antibody (G8.8) |
| CaCl2 1M solution | BioVision | B1010 | sterile |
| Cell culture dishes 35 mm | Corning | 430165 | Non-pyrogenic, sterile |
| Collagenase Type I | Wortington | LS004194 | |
| Corn oil | Any avaliable | Any avaliable | From grocery store |
| Corning cell strainer size 70 μm | Sigma-Aldrich | CLS431751-50EA | |
| Digital Stirrer PC-410D | Corning | Item# UX-84302-50 | |
| Dispase II | Sigma-Aldrich | D4693-1G | |
| Dissecting scissors, curved blunt | McKesson Argent | 487350 | Metzenbaum 5-1/2 Inch surgical grade stainless steel non-sterile finger ring handle |
| DNase I | Akron Biotech, catalog number | AK37778-0050 | |
| Dulbecco’s Modified Eagle’s Medium – low glucose (DMEM) | Sigma-Aldrich | D5546-500ML | with 1000mg/L glucose and sodium bicarbonate, without L-glutamine |
| Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) | Millipore | DF-042-B | without HEPES, L-glutamine |
| Easypet 3 pipette controller | Eppendorf | 4430000018 | with 2 membrane filters 0.45 µm, 0.1 – 100 mL |
| Ethanol | Sigma-Aldrich | E7023-500ML | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | E6758 | |
| Fisher Vortex Genie 2 | Fisher Scientific | 12-812 | |
| FlowJo version 10 | Any available | Any available | |
| Fluorescence binocular microscope Axioplan2 | Carl Zeiss | ID# 094207 | |
| Ghost Red 780 Viability Dye | Tonbo Biosciences | 13-0865-T100 | |
| GlutaMAX Supplement | ThermoFisher Scientific, Gibco | 35050061 | |
| Glycerol 99% | Sigma-Aldrich | G-5516 | |
| Hand tally counter | Heathrow Scientific | HEA6594 | |
| Hank's Balanced Salt Solution (HBSS) | Sigma Millipore | H6648-500ML | Modified, with sodium bicarbonate, without calcium chloride, magnesium sulphate, phenol red. |
| Hank's Balanced Salt Solution (HBSS) | ThermoFisher Scientific | 14025092 | With calcium, magnesium, no phenol red. |
| Hausser Bright-Line Phase Hemocytometer | Fisher Scientific | 02-671-51B | 02-671-51B |
| HEPES 1M solution | ThermoFisher Scientific, Gibco | 15630-080 | Dilute 1/10 in ddH20 |
| HyClone Fetal Bovine Serum (FBS) | Fisher Scientific | SH3007002E | |
| Hydrochloric Acid (HCl), 5N Volumetric Solution | JT Baker | 5618-03 | To adjust Tris buffer pH |
| Innova 4230 Refrigerated Benchtop Incubator | New Brunswick Scientific | SKU#: | Shaker; 37 °C, 5% CO2 in air |
| Iris scissors | Aurora Surgical | AS12-021 | Pointed tips, delicate, curved, 9 cm, ring handle |
| Isoflurane Inhalation Anesthetic | Southern Anesthesia Surgical (SAS) | PIR001325-EA | |
| MgCl2 1M solution | Sigma-Aldrich | 63069-100ML | |
| Microcentrifuge tubes 1.5 mL | ThermoFisher Scientific | 3451 | Clear, graduated, sterile |
| Microsoft Power Point | Any available | Any available | |
| NaCl powder | Sigma-Aldrich | S-3014 | |
| Nalgene 25 mm Syringe Filters | Fisher Scientific | 724-2020 | |
| Pen Strep | Gibco | 15140-122 | |
| pH 510 series Benchtop Meter | Oakton | SKU: BZA630092 | |
| Phosphate buffered saline (PBS) | ThermoFisher Scientific | 10010023 | pH 7.4 |
| Pure Ethanol 200 Proof | Pharmco-Aaper | 111000200 | |
| Red blood cell lysis buffer 10x | BioVision | 5831-100 | |
| Roto-torque Heavy Duty Rotator | Cole Parmer | MPN: 7637-01 | |
| Safe-lock round bottom Eppendorf tubes 2 mL | Eppendorf Biopur | 22600044 | PCR inhibitor, pyrogen and RNAse-free |
| Scissors | Office Depot | 375667 | |
| Sorting flow cytometer MoFlo Astrios EQ | Beckman Coulter | B25982 | With Summit 6.3 software |
| Sorvall Legend Micro 17R Microcentrifuge | Thermo Scientific | 75002441 | All centrifugation performed at RT |
| Sorvall RT7 Plus Benchtop Refrigerated Centrifuge | Thermo Scientific | ID# 21550 | RTH-750 Rotor. All centrifugation performed at RT |
| Stemi SV6 stereo dissecting microscope | Carl Zeiss | 455054SV6 | With transmitted light base |
| Tamoxifen | Millipore Sigma | T5648-1G | |
| Trizma base powder | Sigma-Aldrich | T1503 | |
| Trypan blue solution | Millipore Sigma | T8154 | |
| Two Dumont tweezers #5 | World Precision Instruments | 500342 | 11 cm, Straight, 0.1 x 0.06 mm tips |
| Upright microscope | Any available | Any available | With transmitted light base |
| Vacuum filtration systems, standard line | VWR | 10040-436 | |
| Variable volume micropipettes | Any available | Any available |
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Citer Cet Article
Zyrianova, T., Basova, L. V., Makarenkova, H. Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands. J. Vis. Exp. (148), e59602, doi:10.3791/59602 (2019).