肺动脉平滑肌细胞从新生小鼠隔离
Summary
我们已经开发了一种新的和可重复的技术来隔离为P7作为年轻的小鼠原代培养的肺动脉平滑肌细胞（PASMC），从而使更好地参与新生儿平滑肌细胞的收缩和舒张的信号转导通路的研究。
Abstract
肺动脉高压是在婴幼儿的发病率和死亡率的一个重要原因。在历史上，一直存在重大研究信号通路参与血管平滑肌收缩的肺动脉平滑肌细胞从胎羊。虽然羊长期肺动脉高压的一个很好的模型，他们都非常昂贵，缺乏发现在小鼠遗传操作的优势。相反，该系统是一个重大的限制无法从小鼠分离的PASMC。在这里，我们描述了的的小鼠肺动脉平滑肌细胞的原代培养的隔离P7，P14，和P21小鼠使用先前所描述的技术，Marshall 等的变形例26中 ，曾用于隔离大鼠PASMC。这些鼠肺动脉平滑肌细胞代表了一种新的工具在新生儿期的信号转导途径的研究。简单地说，淤浆的0.5％（重量/体积）的琼脂糖+通过右心室（RV）的M199培养基中的0.5％的铁粒子注入肺血管床。该铁颗粒是直径为0.2μM，并不能穿过肺毛细血管床。因此，铁肺小动脉（PA）递交。琼脂糖，取出游离肺膨胀。含铁船舶用磁铁被拉下来。胶原酶（80单位/毫升）处理，并进一步解离后，将容器放入一个组织培养皿中的M199培养基含有20％胎牛血清（FBS）和抗生素（M199完全培养基）中，以允许到培养皿中的细胞迁移。初始板的细胞，成纤维细胞和肺动脉平滑肌细胞是50-50混合物。因此，下拉过程重复多次，以实现更纯净的的PASMC人口和消除任何残余的铁。平滑肌细胞的身份被确认通过免疫组化平滑肌肌球蛋白和结。
Introduction
肺动脉高压是正常期间宫内寿命时间作为气体交换的主要器官，胎盘在子宫内的心输出量只有10％被循环通过肺血管床。类似全身的压力，由于肺血管阻力升高，肺动脉压力。随着妊娠的进展，有快速增长的小PA在肺部，准备对胎儿的肺血流量的急剧增加，发生在出生1。当正常的围产期过渡失败，在近期和足月，其结果是持续性肺动脉高压（PPHN）的新生儿。 PPHN是由许多不同的相关病症引起的一组临床综合征。然而，所有这些婴儿有着共同的病理生理功能，如肺血管阻力升高，低氧血症，由右至左分流血流跨持续胎儿连接，如动脉导管未闭或阿门未闭。 PPHN每千名活产和影响2-6，传达的死亡率，以及显着的短期和长期患病2 8-10％的风险。此外，极低出生体重早产儿可能会由于其潜在的肺部疾病发展肺动脉高压。最常见的潜在肺疾病的早产儿支气管肺发育不良（BPD）。虽然BPD的整体风险与胎龄和出生体重，目前还不清楚为什么一个子集，这些婴儿的发展显着的肺动脉高压，以及如何适当地对待这些婴儿。差的结果，包括住院时间延长，死亡率增加，是常见的3-6。
从历史上看，从健康动物绵羊胎儿肺动脉平滑肌细胞或猪胎儿肺动脉平滑肌细胞已被用来研究信号通路参与在正常出生后肺血管过渡。这些通常是孤立的第五代性PAÑ 绵羊或猪胎儿的交付实施安乐死之前，任何自发呼吸7-9。此外，一些研究者已经分离并利用PASMC从年龄稍大的自主呼吸的羔羊和仔猪在3天，2周，4周10-12。最近，一些团体已经成功地分离和利用PASMC孤立羔羊PPHN检查紊乱信号通路在疾病状态13-17。这些细胞已被证明是一个有价值的工具来检查信号通路是至关重要的正常和病变的近期和长期的肺血管。然而，他们不给洞察信号通路的影响，早产儿肺动脉高压。他们也不可以看出，在小鼠疾病模型的遗传操作的可能性。
长期以来，大鼠和小鼠模型已被用于模型BPD以及最近正被用于模型肺HYpertension造成BPD 18-22。新生大鼠是诱人的工作，由于其较大的规模，但他们也遭受缺乏基因改造的潜力。转基因动物已被广泛用于研究特定基因的目标，整个动物生理学新生小鼠的影响，但至今没有任何人曾成功分离PASMC从这些小老鼠。通过隔离PASMC，可以得到更多的信息，关于如何在应对环境刺激和/或遗传修饰，特别是在肺动脉平滑肌途径改变。此外，现场PASMC可以成像实时的，，检查快速变化的关键信号分子，如钙和活性氧23-25。最近，我们描述了使用的变化的技术，Marshall 等从成年小鼠肺动脉平滑肌细胞成功地分离出26用于隔离大鼠肺动脉平滑肌细胞23,25,26。我们现在已经适应了ND扩展这种技术小老鼠7-21日龄（P7，P14，P21）。到这个新的PASMC隔离技术的主要限制是，它要求多鼠标，以产生足够的用于实验的细胞，细胞的生长非常缓慢，这是主平滑肌细胞的特征。尽管有这些限制，我们相信这种技术来隔离新生小鼠PASMC将允许增强调查的关键信号通路参与了肺动脉高压的发展，并表示在这一领域的一个显着的进步。
Protocol
Representative Results
Discussion
在这个手稿中，我们描述了在第一次的PASMC在P7，P14，和P21小鼠的隔离。为了做到这一点，通过琼脂糖凝胶和0.2μM铁颗粒的浆料注入到PA的RV。由于小尺寸的铁粒子，它们无法通过肺毛细血管床，因此沉积在小PA。肺部膨胀，除去游离。含铁的容器内被拉出使用磁铁的溶液。最终，该船舶被镀成的组织培养皿中，细胞迁移的船只，到培养板。的初始板的细胞是成纤维细胞和肺动脉平滑肌细胞的混合物…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院HL109478（KNF）的支持。作者吉娜·金正日和乔安·泰勒承认并感谢他们的协助隔离和保持PASMC文化。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Bard Parker surgical blade handle | BD | 371030 | |
| Stainless steel surgical blades #10 (sterile) | Miltex | 4-310 | |
| Syringes (3 ml and 5 ml, sterile) | BD | 309657 and 306646 | |
| Needles (27 G, sterile) | BD | 305109 | |
| Angiocatheter (24 G, sterile) | BD | 381412 | |
| Monoject blunt cannula (15 G) | Kendall | SWD202314Z | |
| Sutures | Fisher Scientific | NC9782896 | |
| Dynal magnet particle collector | Invitrogen | 120-01D | This is a critical tool for the protocol. |
| Tissue culture plates (35 mm, 60 mm, and 10 cm, sterile) | BD | 353001, 353004, and 353003 | Any brand of tissue culture plates will be fine. |
| Iris Scissors (4 ½ inch stainless steel) | American Diagnostic Corporation | 3424 | |
| Forceps (4 inch stainless steel) | Quick Medical | L5-5004 | |
| D-PBS | Mediatech | 21-031-CV | |
| M199 media | Mediatech | 10-060-CV | |
| Penicillin/streptomycin | VWR | TX16777-164NWU | |
| Fetal bovine serum | Hyclone/Thermo Scientific | SH3091003 | Heat inactivate at 55 °C for 45 min. For consistency in results, lot match all serum and obtain from same vendor. |
| Iron particles (iron (II, III) oxide powder) | Aldrich Chemical Company | #31,006-9 | |
| Agarose | Sigma | A9539 | |
| Collagenase (made to 80 U/ml) | Sigma | C5138 | |
| Isoflurane | Butlet Schein | NDC 11695-6776-1 | |
| Nikon Eclipse TE2000-U with a Cascade Photometrics 12-bit camera | Nikon | TE2000-U | Any good light microscope will be fine to observe PASMC in culture. |
| Anti-desmin antibody | Sigma | D-8281 | Use at 1:200 dilution for immunostaining. |
| Anti-smooth muscle myosin | Biomedical Technologies | BT-562 | Use at 1:2,000 dilution for immunostaining. |
| Rhodamine-red anti-rabbit secondary | Molecular Probes/Invitrogen | R-6394 | Use at 1:200 dilution for immunostaining. |
| Nikon Eclipse TE-300 fluorescent microscope and Cool Snap digital camera | Nikon | TE300 | Any good epifluorescence microscope will be fine for immunostaining. |
| Cyclic nucleotide phosphodiesterase assay kit | Enzo Life Sciences | BML-AK800-0001 | This is the only colorimetric PDE enzyme activity assay available. |
| Sildenafil | Sigma | PZ-0003 | A PDE5-selective inhibitor is required for the PDE enzyme activity to determine specificity of cGMP hydrolysis. |
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