CD146的分离<sup> +</sup>从大鼠肺组织驻地肺间质基质细胞
1Sinclair Centre for Regenerative Medicine,Ottawa Hospital Research Institute, 2University of Ottawa, 3Department of Neonatology and Pediatric Critical Care Medicine, Medical Faculty and University Hospital Carl Gustav Carus,Technische Universität Dresden, 4DFG Research Center and Cluster of Excellence for Regenerative Therapies (CRTD),Technische Universität, Dresden, 5Children’s Hospital of Eastern Ontario Research Institute
Summary
这个协议描述了从大鼠获得原发性肺癌驻地间充质基质细胞，通过使用酶消化，密度梯度分离，塑料吸附和CD146 +磁珠选择的隔离技术。
Abstract
间充质基质细胞（MSC）越来越多地认识到它们的治疗潜力在很宽范围的疾病，包括肺疾病。除了使用骨髓和外源细胞疗法脐带干细胞的，还增加居民组织的MSCs在修兴趣和再生潜能。此外，它们可能在正常器官发育的作用，并已被归因于疾病的作用，特别是那些患有纤维变性性质。这些居民组织干细胞研究中的主要障碍是缺乏对这些细胞的分离和鉴定明确的标记。这里所描述的隔离技术应用于肺间充质干居民（L-MSCS）的多个特征。一旦老鼠的牺牲，肺被删除，漂洗多次，以去除血。继手术刀机械分离，肺部使用I型胶原酶，中性蛋白酶和DNA酶I型的obtaine混合消化2-3小时D单细胞悬浮液，随后洗涤并铺在密度梯度介质（密度1.073克/毫升）中。离心后，从相间洗涤细胞并涂布在培养处理的烧瓶中。细胞是4-7天在生理5％O 2，5％CO 2的条件下培养。耗尽成纤维细胞（CD146 – ），并确保只有L-干细胞（CD146 +），CD146为+细胞阳性选择的人口通过磁珠选择执行。总之，这个过程可靠地产生主L-MSC的进一步的体外研究和操纵的群体。因为协议的性质，它可以很容易地转换为其他实验动物模型。
Introduction
间充质基质细胞（MSC）越来越多地认识到它们的治疗潜力在很宽范围的疾病，包括肺疾病。除了使用骨髓和外源细胞疗法脐带干细胞的，还增加居民组织的MSCs在修兴趣和再生潜能。在开发过程中，包括肺，间质发育是线索的重要来源，而居民干细胞是可能的候选人是在这个中心。此外，有证据表明居民的MSCs在成人疾病扰动，包括癌症1,2和纤维化3。这些居民组织干细胞研究中的主要障碍是缺乏这些细胞4的分离和鉴定明确的标记。干细胞抗原-1（SCA-1）在小鼠中作为用于各种组织的干细胞的标志物被鉴定，并且可用于L-的MSC 5的隔离，但不幸的是没有已知的其他物种6直向同源物。研究人员已经报道了各种不同的分离方法对L- MSC的从任肺组织或流体的分离。 – / CD45 – – / CD90 +细胞 7，CD31 – / CD45 – /上皮细胞粘附分子（EpCAM的） -这些从荧光激活细胞分选（FACS）为基础的方法选择用于CD31变化/的Sca-1 +细胞8，多药耐药转运ATP结合盒G（ABCG2）阳性细胞9或赫斯特33342染料外排10，塑料粘附11,12和迁移出组织糜13。
在这里所提出的方法的优点是数倍。通过使用温和酶消化和密度梯度14中，可以得到包含的MSC，但不包括上皮或内皮细胞的密度范围的所有细胞。随后塑料吸附步骤确保只有mesenchymal细胞粘附和留在文化，消除白细胞。最重要的是然而，CD146 +选择步骤允许消除成纤维细胞，因为这些细胞不表达CD146。细胞粘附分子，CD146的表达是积极与多能相关，并且因此是一个很好的标志物从间充质细胞群体15-19剔除成纤维细胞。这是通过使用CD90作为选择标记的优点，因为它不仅在干细胞，而且在lipofibroblasts 5,20表示。在这个协议中，我们已经明确选择磁珠选择，因为它是在细胞上温和，而且整个过程可在无菌条件下进行。相对于生长方法这种隔离方法的另一个重要优点是，它是比较快的，6-10天，而不是在一个月或更长时间的生长方法。初始分离后三到五天的间充质人口准备CD146 +塞莱 ction;后3到5天，CD146 +细胞准备用于实验或可以冷冻供以后使用。培养的下降时间，从而提高了电池的质量的MSC朝向延长体外培养 19成纤维细胞转分化。最后，由于该协议的性质，有可能通过简单地调节消化酶和孵育时间的选择选择合适的抗体，或甚至其他器官系统这种方法适用于其它物种。
这种隔离方法的详细方案在下面给出， 并在图1A和1B分别设置在隔离和CD146 +亚群的随后选择的示意图。此外，详细信息包含用于传代，冷冻和解冻这些细胞。
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图1.肺间质细胞（A）和随后的CD146 +细胞的选择（B）的最小=分钟的隔离的示意图概览; EDTA =乙二胺四乙酸; 第二抗体=二级抗体; α-CD146抗体=初级抗CD146抗体; -ve细胞= CD146阴性细胞; +已经细胞= CD146阳性细胞。 请点击此处查看该图的放大版本。
Protocol
所有的程序是由渥太华大学的动物护理委员会（动物伦理协议OHRI-1696）的批准。动物保健是按照机构的指导进行。 1.肺间质干细胞的分离在50毫升管准备酶混合物：权衡30单位中性蛋白酶2500ü我胶原酶和500üDNA酶一，这些款项足以满足成年小鼠或大鼠小狗的肺部。在4℃直至使用上隔离和存储的天制备。 牺牲幼鼠第13天通过腹膜内注射戊巴比妥钠（0.2毫升，65毫克/…
Representative Results
间充质干的，密度和贴壁的最可靠的物理特性二，在这个协议的第一部分是用来获取包含L-干细胞肺的间质细胞部分。虽然密度梯度相间将包括除了肺间质细胞的单核细胞和巨噬细胞，塑料粘附然后培养3-5天确保只有肺间质细胞保持。确实该细胞群表达了经典的MSC表面标记CD73，CD90和CD146，并为负标记CD34，CD45，主要组织相容性复合物II类（MHCII）-RT1B，CD11b和CD79A，表明不再?…
Discussion
主要L-干细胞的分离和培养提供了一个机会，以更好地了解它们的功能和它们与其他细胞群在细胞水平上的互动，以及它们在肺的发育，健康和疾病的作用。这是特别重要的，因为在缺乏这些细胞的特定单个标记的使得它几乎不可能来研究这些细胞中的原位 。与所有主要的细胞群，应该记住，这些细胞更可能改变它们的性质较长它们保持在培养物19（在24中综述）。保持左?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
JJPC is supported by a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship. MAM receives a merit scholarship from the German National Academic Foundation – Studienstiftung des Deutschen Volkes and is supported by a grant from the EFCNI (European Foundation for the Care of the Newborn Infant). BT is supported by CIHR, the Canadian Lung Association, the Stem Cell Network and the Children’s Hospital of Eastern Ontario Research Institute.
Materials
| Neutral Protease | Worthington Biochemical Corporation | LS02104 | Prepare on day of isolation and store at 4°C until use |
| Collagenase I | Worthington Biochemical Corporation | LS004196 | Prepare on day of isolation and store at 4°C until use |
| DNAse I | Sigma-Aldrich | D5025 | Prepare on day of isolation and store at 4°C until use |
| Pentobarbital sodium (Euthanyl) | Bimeda-MTC Animal Health Inc, Dublin, Ireland | – | Use 0.2 mL for rat pups between 20-30g; larger animals may need more. |
| Dulbecco’s PBS + Sodium-pyruvate + Glucose | Life Technologies | 14287-072 | Very crucial to use this type of D-PBS, as the calcium and magnesium that are present in this D-PBS facilitate facilitate the enzymatic digestion |
| Ficoll-Paque PREMIUM (1.073 g/cm3) | GE Healthcare | 17-5446-52 | Density gradient media. To obtain a good interphase, it is crucial that the layering of the single cell suspension occurs at a >45o angle at very low speed, and that the subsequent centrifugation is done at 19oC. |
| αMEM | Sigma-Aldrich | M8042 | Warm in 37oC water bath before use |
| 200 mM L-Glutamine | Life Technologies | 25030-164 | |
| 100x Antibiotic-Antimycotic | Life Technologies | 15240-062 | Penicillin/Streptomycin/Fungizone |
| M-280 Dynabeads | Life Technologies | 11205D | Magnetic beads |
| biotinylated polyclonal rabbit-anti-mouse IgG | Dako, Agilent Technologies | E0464 | Biotinylated secondary antibody that matches with the anti-rat CD146 antibody described below |
| DynaMag5-magnet | Life Technologies | 12303D | Magnet that is recommended for use with Dynabeads. NOTE: magnet should be chosen based on the manufacturer’s instructions of the magnet beads of choice. |
| TrypLE express | Life Technologies | 12605-028 | Gentle non-trypsin alternative, use 1 mL of TrypLE express/ 25cm2 surface area. After detachment, 10 mL L-MSC culture medium is sufficient to inactivate 3 mL TrypLE |
| anti-rat CD146 antibody | Lifespan Biosciences Inc. | C35841 | 12.5 μl per 0.5 x 106 cells |
| Pentaspan (pentastarch solution) | Bristol-Myers Squibb Canada | – | Can be obtained through local blood donation services or hematology departments. Alternatively, one could use the PSI 20% Pentastarch solution (Preservation Solutions, PST001) diluted 1:1 with 0.9% NaCl. |
| Mr.Frosty freezing container | ThermoFisher Scientific | 5100-0001 | Improves viability when freezing L-MSCs overnight at -80oC |
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Citer Cet Article
Collins, J. J., Möbius, M. A., Thébaud, B. Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs. J. Vis. Exp. (112), e53782, doi:10.3791/53782 (2016).