粘附细胞用惰性气体喷射的透
Summary
这个协议描述了用于贴壁细胞用惰性气体喷射的暂时通透性的方法。这种技术有利于遗传物质和生物分子转移到贴壁哺乳动物细胞中通过机械力的利用来破坏细胞膜。
Abstract
各种细胞的转染技术存在并且这些可以细分到三个大类：病毒，化学和机械。这个协议描述了一种机械方法，用惰性气体喷射，可以方便的通常不可渗透大分子转移到细胞中，以时间上通透贴壁细胞。我们相信这种技术的工作原理是赋予对贴壁细胞的质膜的剪切力，从而在临时形成微孔。一旦创建了这些孔，然后将细胞可渗透的遗传物质和其它生物分子。所涉及的机械力，还是从他们的衬底运行永久损坏或脱落细胞的风险。有，因此，一个狭窄的范围内的惰性气体动力学，其中该技术是有效的。惰性气体喷流在不同的透贴壁细胞系包括HeLa细胞，HEK293和人的腹主动脉内皮细胞被证明有效。这个协议是适当的在p在体外和，贴壁细胞ermeabilization因为我们已经证明， 在体内 ，显示出它可以在今后的临床应用中用于研究和潜在的。它也有一个空间限制的方式透化细胞的优势，这可能被证明是一个有价值的研究工具。
Introduction
随着生物医学的发展和细胞力学的理解，传递生物分子进入细胞已成为许多研究领域和药物治疗是至关重要的。不同的技术已被开发，通过细胞膜，进入细胞胞质溶胶中引入外来分子。这些通常可分类为：病毒，化学的或机械的技术。病毒的技术能够转染遗传物质，例如使用病毒载体1的RNA和DNA。病毒的技术往往具有高的效率在某些细胞系中，但是它们有用于免疫和炎症反应2的潜力。常见的化学转染方法包括沉淀磷酸钙3，细菌外毒素4和脂质体转染5。这些技术已被证明是有效的，然而，某些问题时，如细胞毒性和非特异性。此外，如钙phosphat技术ë沉淀已发现具有转染效率可达70％，但仅在某些细胞系中，很少在原代细胞6。这是值得注意的是越来越多的研究工作正在投入的原代细胞，特别是在临床使用和DNA功能的研究设计各种处理的情况下。
通过机械刺激细胞膜瞬时中断是用于将外来分子进入细胞的一种替代方法。技术包括：使用超声波来破坏细胞膜10-12，粒子轰击就证明了“基因枪”，其拍摄粒子结合有显微注射分子7，利用电场来破坏膜8,9电穿孔，声孔效应基因进入细胞13，以及最近的，已被证明在时间上通透哺乳动物细胞14,15的流体剪切应力的应用。虽然技工人的方法避免上述的一些问题，它们通常伴随着较低的效率和非常复杂的和专门的设置。大气压辉光放电火炬（APGD-T）于麦吉尔开发的功能化表面和体外 16分离贴壁细胞的初始目标。偶然，人们发现，一活/死染色被用来在某种程度上被取入细胞而不被加入渗透剂。此外，这似乎只发生在其中发生在火炬路径匹配井的禁区。调查的APGD-叔的通透能力，以及继续在对照研究中，我们发现，如果没有激发等离子体的载体惰性气体射流也能够透化细胞。这违背了最初的假设，即由等离子体射流产生的活性物种暂时受损的细胞膜功能，并建议只是式机械密封卡尔力量足以引起细胞通透性。从这里，研究持续在我们的实验室，试图量化和表征惰性气体射流的通透性效率以及看看它的转染能力16。
通过这些研究，已经发现，微孔做实际上形式在质膜，并且这些孔隙倾向于在大约5秒15重新密封。我们已经证明了该技术的效用在体外和体内在鸡胚绒毛尿囊膜上。
Protocol
Representative Results
Discussion
惰性气体喷射通透是贴壁细胞转染的有效技术。它提供了传送生物分子进入细胞的使用的机械力，从而省去了潜在的有害化学物质或病毒载体的能力。该技术可以潜在地给研究者和临床医生的效率和相对简单的方法来精确地转染的细胞。透化的选择性也是唯一允许研究人员只是将某些细胞中的单个菌落这可能是在多种应用中是有用的。
一些参数可以为个别的实验，如毛细管?…
Declarações
The authors have nothing to disclose.
Acknowledgements
笔者想感谢以下资金来源：健康研究加拿大学院（CIHR），自然科学和工程研究理事会（NSERC）。
Materials
| Vitality hrGFPII-1 | Agilent Technologies Canada | 240143-57 | Green fluorescent protein for transfection experiments on HeLa cells |
| Dextran, AlexaFluor 488; 10,000 MW, Anionic, Fixable | Life Technologies Inc. | D22910 | dextran for permeabilization experiments |
| LIVE/DEAD Viability/Cytotoxicity Kit | Life Technologies Inc. | L3224 | for counterstaining of mammalian cells |
| Prolong Gold Antifade Reagent | Invitrogen | P36930 | for mounting slides when imaging |
| Ultra High Purity Helium | Praxair Canada Inc. | HE 5.0UH-T | |
| Hyclone DMEM/ High Glucose Media – 500 ml | Thermo Scientific | SH30022.01 | for HeLa cells |
| Fetal Bovine Serum | Invitrogen | 26140079 | For DMEM media |
| Penicillin-Streptomycin, liquid | Invitrogen | 15140-122 | For DMEM media |
| Phosphate-Buffered Saline (PBS) 1x | Produced in lab | ||
| Table 1. List of required reagents | |||
| 6-Well Clear TC-Treated Microplates | Corning | 3506 | |
| #1 22 x 22 Coverslips | Fisher | 12-542B | |
| Glass Capillaries | World Precision Instruments | WPI Sizing Information | |
| ID (mm): 1, 0.86, 0.68, 0.5 Order #: 1B200, 1B150, 1B120, 1B100 | |||
| Mass-Flo Controller | MKS | M100B01314CS1BV | Mass flow controller, Series M100B, requires an external 12 V power supply |
| USB-6009 DAQ Device | National Instruments | 779026-01 | |
| UniSlide Assembly with stepping motor and controller | Velmex | Two series MA15 UniSlide Assemblies, fitted with Vexta 1.8° stepping motors provided by Velmex, and controlled by a VXM Stepping Motor Controller | |
| Table 2. List of required equipment |
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