在胎儿肺细胞的机械传导的实验系统研究
Summary
机械力发挥关键作用，在肺发育和肺损伤。在这里，我们描述了一个隔离啮齿动物胎儿肺Ⅱ型上皮细胞和成纤维细胞，并揭露他们使用的机械刺激的方法<em>在体外</em>系统。
Abstract
在子宫内产生的机械力通过重复呼吸运动和流体腹胀是正常的肺发育的关键。肺发育的一个关键组成部分是分化的肺泡Ⅱ型上皮细胞，肺表面活性物质的主要来源。这些细胞也参与流体的动态平衡，在肺泡腔，宿主防御，损伤修复。此外，远端肺组织细胞，可以直接暴露在机械通气，出生后夸张的拉伸。然而，其中肺细胞感应机械刺激影响肺癌的发展和促进肺损伤的确切的细胞和分子机制尚未完全了解。在这里，我们提供了一个简单的和高纯度的方法来隔离II型细胞和啮齿动物胎肺成纤维细胞。然后，我们描述了在体外系统，Flexcell的应变单位，提供机械刺激胎儿细胞，模拟机械力胎儿肺发育或肺损伤。该实验系统提供了一个极好的工具来暴露伸展胎儿肺细胞的细胞和分子机制研究。使用这种方法，我们的实验室已经确定了几个受体和信号转导蛋白在胎儿肺发育和肺损伤，机械传导参加。
Protocol
1。与ECM蛋白涂层板在无菌条件下，混合冷无菌1X PBS每盘12毫升120微克的粘连。 采取Bioflex未处理板，并添加到每口井的解决方案2毫升（粘连最后的浓度为2微克/厘米2）。另外，其他的ECM蛋白可以使用，如胶原-1 [10微克/厘米2]，纤维连接蛋白[5微克/厘米2]，玻[0.5微克/厘米2]或弹性[10微克/厘米2]。什么是粘连涂层的涂层技术是类似的。确保井完?…
Discussion
在这个手稿中，我们描述了一种方法来隔离胎儿II型上皮细胞和成纤维细胞和揭露他们使用的Flexcell应变仪的机械刺激。我们已经用这种技术来评估上皮细胞的分化1,2和研究舒展3-9激活的受体和信号通路的。此外，这种方法也可以用来研究由10,11机械损伤诱导细胞反应。胶原酶消化肺组织的程序是基于。它需要利用II型细胞的倾向消化后聚集在一起。在执行这一技术的重要?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
支持由NIH资助的HD052670。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| DMEM | Sigma | D5648 | |
| HEPES | Sigma | H3375 | |
| Collagenase 1 | Sigma | C0130 | |
| Collagenase 1A | Sigma | C9891 | |
| Chicken serum | Sigma | C5405 | |
| Screen cups | Sigma | CD1-1KT | |
| Syringe filters | Fisher Scientific | 09-754-25 | |
| 100 micron nylon mesh | Small Parts, INC | CMN-100-D | |
| 30 micron mesh | Small Parts, INC | CMN-30-D | |
| 15 micron mesh | Dynamic Aqua-Supply Ltd. | NTX15 | |
| Laminin | Sigma | L2020 | |
| Collagen-1 | Collagen Biomed | PC0701 | |
| Fibronectin | Sigma | F1141 | |
| Vitronectin | Sigma | V-0132 | |
| Elastin | Sigma | E-6402 | |
| Bioflex plate | Flexcell International | BF-3001U | Uncoated |
| Flexcell Strain Unit | Flexcell International | FX-5000 |
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Tags
Experimental SystemMechanotransductionFetal Lung CellsMechanical ForcesLung DevelopmentAlveolar Type II Epithelial CellsPulmonary SurfactantFluid HomeostasisDistal Lung Parenchyma CellsExaggerated StretchMechanical VentilationMolecular MechanismsCellular MechanismsMechanical StimuliLung InjuryIsolation MethodFlexcell Strain UnitIn Vitro SystemMechanical Stimulation
Cite This Article
Wang, Y., Huang, Z., Nayak, P. S., Sanchez-Esteban, J. An Experimental System to Study Mechanotransduction in Fetal Lung Cells. J. Vis. Exp. (60), e3543, doi:10.3791/3543 (2012).