Name: in vitro method to control concentrations of halogenated gases in cultured alveolar epithelial cells
Uploaded: 2018-10-23T14:30:00
Description: Watch this Scientific Journal Video about In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells at JoVE.com

作者承认, 奥弗涅区域理事会 ("新 Chercheur 一个涅" 2013) 和法国法新社谜和 séjour de 护理 ("方案 de 谜 Translationnelle en 桑特"ANR-13-PRTS-0010) 的赠款。出资人对研究设计、行为、分析或本文的编写没有影响。

1. 肺泡上皮细胞培养 (hAELVi)
<ol><li>解冻<ol>
<li>移液器4毫升的培养现成使用的人肺泡上皮 (huAEC) 培养基在一个15毫升塑料管和快速解冻的小瓶在预热水浴 (37 摄氏度)。</li>
		<li>将解冻后的细胞悬浮液转移到含有4毫升培养基的15毫升塑料管上, 然后离心管在 200 x g 处进行5分钟。</li>
		<li>吸出上清液, 用5毫升培养培养基重悬细胞颗粒。然后, 将细胞转移到 T25 烧瓶中。</li>
		<li>在标准条件下培养细胞 (5%...

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunomodulatory+effect+of+sevoflurane+in+endotoxin-injured+alveolar+epithelial+cells.">The immunomodulatory effect of sevoflurane in endotoxin-injured alveolar epithelial cells.</a> Anesthesia and Analgesia. (3), 638-645 (2007).</li><li>Schl&#228;pfer, M., Leutert, A. C., Voigtsberger, S., Lachmann, R. A., Booy, C., Beck-Schimmer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sevoflurane+reduces+severity+of+acute+lung+injury+possibly+by+impairing+formation+of+alveolar+oedema.">Sevoflurane reduces severity of acute lung injury possibly by impairing formation of alveolar oedema.</a> Clinical and Experimental Immunology. (1), 125-134 (2012).</li><li>Nickalls, R. W. D., Mapleson, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+iso-MAC+charts+for+isoflurane,+sevoflurane+and+desflurane+in+man.">Age-related iso-MAC charts for isoflurane, sevoflurane and desflurane in man.</a> British Journal of Anaesthesia. 91 (2), 170-174 (2003).</li><li>Bourdeaux, D., Sautou-Miranda, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+assay+of+plasma+sevoflurane+and+its+metabolite+hexafluoroisopropanol+by+headspace+GC-MS.">Simple assay of plasma sevoflurane and its metabolite hexafluoroisopropanol by headspace GC-MS.</a> Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 878 (1), 45-50 (2010).</li><li>Eger, E. I., Saidman, L. J., Brandstater, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimum+alveolar+anesthetic+concentration.">Minimum alveolar anesthetic concentration.</a> Anesthesiology. 26 (6), 756-763 (1965).</li><li>Perbet, S., Fernandez-Canal, C., Pereira, B., Cardot, J. M., Bazin, J. E., Constantin, J. 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Lung Cellular and Molecular Physiology. 306 (11), L975-L985 (2014).</li></ol>

我们的协议描述了一种简单的方法, 将细胞暴露在卤代麻醉剂的精确部分, 如七氟醚或异氟醚。此外, 我们在这里首次报告-在培养基本身中, 气体馏分与七氟醚和异氟醚浓度之间存在严格的相关性。这一基本步骤现在允许我们安全地使用我们的气密室来研究这些卤代剂在培养的人肺泡上皮细胞中的作用。
目前, 研究七氟醚在肺泡细胞中的作用的大多数科研小组使用的是先用卤化...

急性呼吸窘迫综合征 (ARDS) 是一种以弥漫性肺泡损伤、肺水肿、低氧性呼吸衰竭为特征的临床综合征。虽然 ARDS 代表10% 以上的重症监护病房 (icu) 招生和近25% 的 ICU 患者需要机械通气, 但它仍然是一个未被认可的挑战, 临床医生, 医院死亡率为 35-45%1。尽管进行了大量的研究, 但目前还没有发现有效的 ARDS 药物治疗或预防的方法。两个主要特征导致 ARDS 死亡率: 肺泡液清除 (AFC) 受损 (即肺泡水肿液从远端肺空域的改变吸收) 和严重炎症2。由于 ARDS 死亡率仍然很高, 目前的倡议还应包括初级预防;然而, 一个关键的挑战是找出高危患者, 其中 ards 可能发展和谁将受益, 如果 ards 被阻止。
挥发性卤化麻醉剂, 如七氟醚和异氟醚, 广泛用于在手术室内提供全身麻醉。在全球范围内, 每年有超过2.3亿名患者进行大手术, 需要全身麻醉和机械通气3, 术后肺部并发症对临床结果和医疗保健使用产生不利影响4.使用七氟醚代替异丙酚与改善胸部手术患者的肺部炎症有关, 并显著减少不良事件, 如 ARDS 和术后肺部并发症5。同样地, 异氟醚预处理对 ARDS6、7实验动物模型的呼吸力学、氧合和血流动力学有保护作用。虽然进一步的研究有必要解决吸入剂对非心脏手术结果的影响, 但最近在 meta 分析中观察到肺部并发症的类似减少, 表明吸入麻醉剂-作为反对静脉麻醉-与心脏手术死亡率的降低显著相关8。
关于使用挥发性药物用于 ICU 患者镇静预防或治疗肺部损伤的具体未来数据缺乏。然而, 现在有几个试验支持吸入七氟醚治疗 ICU 患者镇静剂的功效和安全性, 临床前研究表明, 吸入七氟醚和异氟醚7,9改善气体交换, 减少肺泡水肿, 并减弱炎症在 ARDS 的实验模型。此外, 七氟醚减轻 II. 型上皮细胞损伤10, 而异氟醚通过对紧密结蛋白11的调制维持肺泡毛细血管屏障的完整性。然而, 需要进行进一步的研究, 以验证吸入七氟醚和异氟醚的器官保护的实验证据在多大程度上可以转化为人类。本组第一个单中心随机对照试验 (RCT) 发现, 在 ARDS 患者早期使用吸入七氟醚与改善氧合、降低某些亲炎标志物水平和减少肺上皮有关由血浆和肺泡液中晚期糖基化终产物 (sRAGE) 受体可溶性形式的水平评定的损伤12。
综合考虑, 七氟醚和异氟醚对肺损伤的有益作用可能指向依赖于愤怒通路的多种生物通路或功能过程, 即肺泡流体清除 (AFC)、上皮损伤、核因子 (NF)-nf-κb 和巨噬细胞活化的易位。此外, 七氟醚可能影响愤怒蛋白本身的表达。自从我们的研究团队和其他人以前的研究支持在 ARDS 的肺泡炎症和肺上皮损伤/修复的关键作用, 我们设计了一个实验模型, 以提供对机制的翻译理解七氟醚在肺损伤和修复13,14,15。本文研究了一种新型的人肺泡上皮原代细胞系中七氟醚和异氟醚的体外作用, 目的是探讨外周肺 hAELVi (人肺泡上皮慢病毒) 的气-血屏障。永生化), 具有肺泡型 I 类特征, 包括功能性紧结16。
在准备我们的体外调查的设计 (例如,在空气-液体界面与暴露于 "吸入" 七氟醚或异氟醚的肺泡上皮细胞的文化, 我们理解从以前发表的研究,七氟醚的馏分仅在 "空气" 界面17、18、19使用标准监测器 (类似于临床设置中使用的) 进行评估。卤化剂浓度通常根据最低肺泡浓度 (MAC) 值 (例如,在人类中, 对于七氟醚, 0.5, 1.1 和 2.2 vol%, 分别代表0.25、0.5 和 1 MAC) 进行选择; 对于异氟醚、0.6、0.8 和1.3 vol% 分别代表0.25、0.5 和 1 MAC)20。事实上, 七氟醚和异氟醚浓度从未在培养基本身中进行过研究, 因此限制了以往实验模型/仪器的有效性。此外, 大多数实验都使用了在含有七氟醚的空气混合物被冲洗进去后密封的厌氧罐。由于我们的目标是研究肺泡上皮细胞在 "生理" 条件下, 我们认为这种厌氧状态可能不是最佳的, 不会与长期的实验时间相容。因此, 我们开发了自己的系统, 在空气-液体界面培养细胞, 并将它们暴露给卤代剂 (七氟醚和异氟醚), 目的是为这些制剂提供精确控制的 "空气" 组分和 "培养基" 浓度。在我们看来, 这一实验步骤, 这是尚未报告迄今在文献中, 是强制性之前, 任何进一步的体外调查七氟醚和异氟醚。

<table>
	<tbody>
		<tr>
			<td>Sevoflurane</td>
			<td>Baxter</td>
			<td></td>
			<td>Performing experiments using sevoflurane or isoflurane while being pregnant should be strongly discouraged</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Virbac</td>
			<td></td>
			<td>Performing experiments using sevoflurane or isoflurane while being pregnant should be strongly discouraged</td>
		</tr>
		<tr>
			<td>Human Alveolar Epithelial cells</td>
			<td>InScreenex</td>
			<td>INS-CI-1015</td>
			<td></td>
		</tr>
		<tr>
			<td>huAEC Medium (ready-to-use)</td>
			<td>InScreenex</td>
			<td>INS-ME-1013-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic machine circuit</td>
			<td>Drager</td>
			<td>Fabius</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas analyzer</td>
			<td>Drageer</td>
			<td>Vamos Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic gas filter</td>
			<td>SedanaMedical</td>
			<td>FlurAbsord</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Humifier</td>
			<td>Fisher&#38;Paykel</td>
			<td>MR850</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber</td>
			<td>Curver</td>
			<td>00012-416-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatography coupled with mass detection</td>
			<td>Thermo Fisher Scientific, San Jose, CA, USA</td>
			<td>Trace 1310 with TSQ 8000evo</td>
			<td></td>
		</tr>
		<tr>
			<td>Fused-silica column (30 m x 1.4 &#181;m, 0.25 mm ID)</td>
			<td>Restek, Lisses, France</td>
			<td>Rxi-624Sil MS</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro method to control concentrations of halogenated gases in cultured alveolar epithelial cells

急性呼吸窘迫综合征 (ARDS) 是一种弥漫性肺泡损伤, 肺泡液清除和严重炎症的综合征。使用卤代剂, 如七氟醚或异氟醚, 用于重症监护病房 (ICU) 的镇静, 可以改善气体交换, 减少肺泡水肿, 并在 ARDS 期间减弱炎症。然而, 在 ICU 中使用吸入剂进行持续镇静治疗或预防肺部损伤的数据缺乏。为了研究卤代剂在 "生理" 条件下对肺泡上皮细胞的影响, 我们描述了一种在气液界面培养细胞的简单系统, 并将其暴露在卤化剂中, 以提供精确控制的 "空气" 组分和这些制剂的 "中等" 浓度。我们开发了一个密封的气密室, 其中, 人肺泡上皮永生化细胞的板块可以通过麻醉机电路提供的连续气体流暴露于七氟醚或异氟醚的精确、控制的部分。细胞被暴露在4% 七氟醚和1% 的异氟醚24小时。采用气传质谱法测定了溶于培养基中的卤代剂浓度。在第一个小时后, 在培养基中的七氟醚和异氟醚的浓度分别为251毫克/升和25毫克/升。在培养基中溶解的七氟醚和异氟醚的浓度曲线显示了类似的课程随着时间的推移, 一个高原达到在一个小时后暴露。
本协议是专为达到精确和控制的七氟醚或异氟醚在体外的浓度, 以提高我们对在 ARDS 期间上皮性肺损伤的机制的理解, 并测试新的治疗综合 征。

表 1和表 2分别报告了七氟醚和异氟醚在介质中随时间溶解的浓度。
在培养基中, 七氟醚和异氟醚浓度的变化与时间相似。在所需的卤代剂浓度设定后, 在第一个小时内浓度上升。然后到达一个高原, 一直持续到卤化剂的管理停止。在管理中断后, 浓度在一小时内减少 (图 3)。
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Watch this Scientific Journal Video about In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells at JoVE.com

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

我们描述了一个简单的协议, 专门设计, 以达到精确和控制的浓度七氟醚或异氟醚在体外, 以提高我们对介入的机制在上皮性肺损伤的理解和测试小说急性呼吸窘迫综合征的治疗。

体外培养肺泡上皮细胞中卤代气体浓度的控制方法

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...

该方法有助于回答肺损伤和修复研究领域的一些关键问题，如检测卤素辅助作为急性呼吸窘迫综合征的潜在新疗法。这种技术的主要优点是，它依靠一种非常简单的方法培养细胞，并把它们暴露在卤化剂中，以达到精确和受控的浓度。要建造气密室，请使用容量为 6.5 升的密封聚丙烯盒。在箱体侧壁底部钻一个直径为 2.5 厘米的孔。然后插入一个带绿色标记的波纹管，作为气体空气混合物输入管。用硅胶密封。接下来，在对面侧壁的顶部钻一个直径为 2.5 厘米的第二个孔。插入另一个带红色标记的波纹管，并将其与木炭过滤器连接，该滤芯将作为气体空气混合物输出管。然后用硅胶密封。随后，在墙的中心钻一个直径为四毫米的紧孔。插入短输液管，用旋转的雄性诱饵锁在歧管上连接。将其插入气体分析仪，然后用硅胶密封。然后将数字温度计放在气密室内。在实验室抽油机罩下，定制麻醉机电路，通过二氧化碳切换一氧化二氮气体管路。将加热加湿器插入麻醉机和气密室之间的管道中，使气流混合物加热至约 37 摄氏度。用绿色标记的波纹管将气密室插入定制的麻醉机回路。接下来，将气密室放在设置为 37 摄氏度的热板上。将含有人类至上细胞的六井板放入气密室并密封盖子。调节气体流速，快速获得5%的CO2。然后打开卤化剂蒸发器并调整所需百分比。达到目标值后，将新鲜气体流量降低至每分钟 1 升。只要实验需要，气密室就可以保持这种气体流速。随着时间的推移，介质中sevoflurane和异氟兰浓度的相向情况相似。卤化剂的所需浓度确定后，浓度立即上升超过第一小时。然后到达一个高原，一直持续到卤化剂的管理停止。管理中断后，浓度在一小时内降低。此处显示的气体分析仪测量的气密室中卤化剂的分数。这个过程相对便宜，而且很容易采用，即使研究人员以前从未操纵过气密室。按照这个程序，可以执行其他方法，如蛋白质和RNA定量，免疫荧光，蛋白质渗透性和自由运输的测量，以回答有关卤化剂在上皮肺损伤期间的具体影响及其分辨率的问题。该技术开发后，可为急性呼吸窘迫综合征领域的研究铺平道路，以进一步研究ARDS期间上皮肺损伤的机制，并测试新疗法。

Research

JoVE Journal

Medicine

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

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科研

医学

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