A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Chastain A. Anderson; Rachael E. Bokota; Andrew E. Majeste; Walter L. Murfee; Shusheng Wang

doi:10.3791/56709

JoVE Journal > Bioengineering

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Bioingegneria

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

Published: January 18, 2018

doi:

10.3791/56709

Chastain A. Anderson, Rachael E. Bokota, Andrew E. Majeste, Walter L. Murfee, Shusheng Wang

¹Cell & Molecular Biology,Tulane University, ²Biomedical Engineering,Tulane University, ³Cell & Molecular Biology & Ophthalmology,Tulane University

Summary

在这里, 我们描述一个可编程的实验室设备, 可用于创建传统卷烟烟雾和电子香烟气溶胶提取物。这种方法为直接比较传统卷烟和电子卷烟提供了一个有用的工具, 是电子卷烟研究中一个容易进入的切入点。

Abstract

电子卷烟是中、高中学生最受欢迎的烟草产品, 是成人中最受欢迎的替代烟草产品。对电子卷烟使用的后果进行高质量、重复性的研究, 对于了解新出现的公共卫生问题和制定基于证据的监管政策至关重要。虽然越来越多的论文讨论电子卷烟, 但在各组的方法上几乎没有一致性, 对结果的共识也很少。在这里, 我们描述一个可编程的实验室设备, 可用于创建传统卷烟烟雾和电子香烟气溶胶提取物。本协议详细说明了该设备的组装和操作, 并演示了在两个示例应用程序中使用生成的提取物: 一个体外细胞活力测定和气相色谱质谱。该方法提供了一种直接比较传统卷烟和电子卷烟的工具, 是电子卷烟研究中一个容易进入的切入点。

Introduction

尽管保健组织作出了集中的努力, 但烟草产品的使用仍然是全世界可预防死亡的主要原因, 其中大多数死亡归因于吸烟的¹。自2003年进入市场以来, 电子卷烟在烟草产品用户中的知名度越来越高。目前, 电子香烟是最流行的替代传统香烟在美国成年人 (〜 5%)²和最流行的尼古丁传递系统中间 (〜 5.3%) 和高中生 (〜 16%)³。如果目前的趋势继续下去, 电子卷烟有望取代传统的香烟, 为子孙后代。然而, 电子卷烟使用的健康后果仍不清楚。

电子卷烟的研究直到 2013年³^,⁴的电子卷烟普及程度迅速增加, 才开始认真。自那时以来, 采用了一些不同的模式来解决其毒性问题。然而, 许多研究的结果是相互矛盾的, 虽然电子卷烟的毒性一般低于传统卷烟, 但目前没有就电子香烟的健康后果达成共识使用⁵^,⁶^,⁷. 我们以前的研究表明, 电子卷烟对血管内皮的毒性要比传统的香烟低得多, 尽管它们有能力引起 DNA 损伤和诱导氧化应激和细胞死亡,⁸.然而, 在我们得出关于电子卷烟使用的健康后果的确切结论之前, 需要进行更多的研究。

由于常规卷烟是可预防的血管疾病的主要病因⁹, 因此对电子卷烟使用的血管健康风险的兴趣与日俱增, 如¹⁰^、¹¹^、¹²。为了研究电子香烟对血管系统的影响, 我们的实验室开发了一个单片机操作的冒烟/vaping 设备 (图 1)⁸。该装置能够在水或有机溶剂中产生传统卷烟烟雾或电子香烟气溶胶的液体萃取物。由于气流由可调式气流调节器和 PBASIC 定时程序的组合控制, 因此该装置可根据任意数量的用户定义的协议来生成提取物。在这里, 我们详细介绍了该设备的组装和操作以及两个潜在的应用:体外细胞活力评估和气相色谱质谱。

图 1: 吸烟/Vaping 设备.为吸烟/vaping 装置的物理装配示意图在香烟或香烟象电子香烟 (电子烟) 配置 (A) 和坦克电子香烟配置 (B)。组件键: 1) 吸入口;2) 初级收藏器;3) 溢流器;4) 傅书礼瓶真空疏水阀;5) 一般开式电磁阀;6) BS1 单片机;7) 气流调节器;8) 510 螺纹电子烟罐底座。请单击此处查看此图的较大版本.

Protocol

1. 装置的装配将100毫升傅书礼烧瓶 (图 1, #4) 固定在钢制圆环架上, 并通过填充50克氯化钙来形成一个真空阱, 作为干燥剂。用橡胶通孔塞子密封烧瓶, 用石蜡膜包裹塞子接合处, 并在孔中运行吸管。使用乙烯基油管, 将吸管从塞子延伸到 t 形交叉软管连接器。使用乙烯基油管, 连接两个器 (图 1, #2 & #3), 并将第二个器的输出连接到 t …

Representative Results

在24小时内, 人类脐静脉内皮细胞暴露于传统的卷烟烟雾提取物 (EAE) 或电子香烟气溶胶提取物 (的), 有一个显着的 (控制与…… 控件 vs. EAE p< 0.01;n = 6) 细胞存活率降低 (图 3A)。提取物产生的膨化轮廓为 2, 2 秒, 55 毫升喷泡每分钟和正常化的基础上的摩尔浓度尼古丁的设备。暴露于500µM 消耗的尼古丁当量的学会极大地减少了活细胞到11.06 ±…

Discussion

本协议最关键的元素是确保设备在每次提取的开始和结束时都是干净的, 并确保所有的密封都保持稳定, 以使气流保持一致。如果设备没有被正确地清洗, 有风险在样品之间运载。此外, 如果设备在长时间内不洁, 凝结气溶胶和干溶剂就会堵塞系统。请注意, 这是正常的, 有一个压力下降时, 膨化一个传统的卷烟和气流表应调整, 以提供所需的气流期间的粉扑, 而不是当设备是拉动房间空气。该方法的?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者感谢杜兰大学细胞和分子生物学系的 Dr. 罗伯特. Dotson 协助编辑杜兰大学化学系的手稿和 Dr. 詹姆斯. 布林协助采用质谱法设计。作者进一步承认杜兰大学细胞和分子生物学系和杜兰大学化学系的支持和使用空间和设备。这项工作得到了烟草产品管理科学研究金和杜兰大学科学与工程学院安德森的支持。

Materials

12 V AC/DC Wall Mount Adaptor	Digi-Key	T1099-P5P-ND
2.2 Ohm Resistors	Digi-Key	A105635-ND	Used in tandem to generate the 4.4 Ohm resistance in Figure 2A
330 Ohm Resistors	Digi-Key	330QBK-ND
510 Threaded Base	NJoy	N/A	Recovered by dismantalling a second generation NJoy electronic cigarette
Acetic Acid, Glacial	Sigma-Aldritch	A6283
Acetone (Chromatography Grade)	Sigma-Aldritch	34850
Basic Stamp Project Board	Digi-Key	27112-ND	This board contains the BS1 Microcontroller, serial adaptor, power switch, and a barrel pin connector for the AC/DC Wall Mount Adaptor
Basic Stamp USB to Serial Adapter	Digi-Key	28030-ND	An optional component to allow the BS1 serial adaptor to communicate through USB
Buchner Flask (Vacuum Flask) 250 mL	VWR	10545-854
Clear Tape	3M	S-9783
Clear Vinyl Tubing, 3/8" ID	Watts	443064
EGM-2 Endothelial Cell Culture Medium	Lonza	CC-3162
Ethanol	Pharmco-Aaper	111000200
Flow Regulator	Dwyer	VFA-23-BV
Gas Chromatograph	Varian	450-GC
Glass Syringe, 10 mL	Sigma-Aldritch	Z314552
Glass Syringe, 10 µL	Hamilton	80300
High Vacuum Silicon Grease	Dow Corning	146355D
Hose Clamp	Precision Brand	35125
Human Umbilical Vein Endothelial Cells	ATCC	PCS-100-013
Mass Spectrometer	Varian	300-MS
Midget Impinger	Chemglass	CG-1820-01
Neutral Red	Sigma-Aldritch	N4638
Paraffin Film	3M	PM-992
Plate Seal Roller	BioRad	MSR0001
Plate Seal; Foil	Thermo	276014
Ring Stand 20"	American Educational Products	7-G15-A
Solenoid Valve (normally open)	US Solid	USS2-00081
Solid State Relay	Digi-Key	CLA279-ND
Stand Clamp	Eisco	CH0688
Syringe Filter, PES, 0.22 um	Millipore	SLGP033RS
Syringe, 10 mL	BD Syringe	309604
Through Hole Stopper, Size 6	VWR	59581-287
Vacuum Pump	KNF Neuberger	N86KTP

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Citazione di questo articolo

Anderson, C. A., Bokota, R. E., Majeste, A. E., Murfee, W. L., Wang, S. A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol. J. Vis. Exp. (131), e56709, doi:10.3791/56709 (2018).