神经元的初步培养体系的研究疱疹病毒潜伏期和活化
1Department of Microbiology,New York University School of Medicine, 2Molecular Neurobiology Program, Skirball Institute for Biomolecular Medicine,New York University School of Medicine, 3Department of Otolaryngology,New York University School of Medicine, 4Department of Cell Biology,New York University School of Medicine, 5Department of Physiology and Neuroscience,New York University School of Medicine, 6Department of Psychiatry,New York University School of Medicine, 7Center for Neural Science,New York University School of Medicine
Summary
协议描述了一种高效和可重复性的模型系统研究单纯疱疹病毒1型（HSV-1）延迟和重新启动。该法采用同质交感神经神经元文化，并允许使用各种工具，包括RNA干扰和重组蛋白表达的病毒神经元相互作用的分子解剖。
Abstract
单纯疱疹病毒1型（HSV-1）建立终身潜伏感染在周边神经元。这种潜在的水库是经常激活事件的源，确保传输和临床疾病作出贡献。目前的抗病毒药物不影响潜在的水库，有没有疫苗。而裂解复制的分子细节是良好的特点，在神经元的延迟控制机制仍然是难以捉摸的。我们目前所知的延迟是来自于体内研究使用小动物模型，并已确定病毒基因的要求和免疫反应的作用是不可或缺的。然而，这是无法区分病毒神经元的关系，从更一般的感染，在活的动物免疫或支持非神经细胞介导的后果的具体影响。此外，动物实验是昂贵，费时，并在有限的可用选项的操作主机流程。为了克服这些限制，只有一个神经元系统的迫切需要，抄录在体内的潜伏期和活化的特点，但在同质化和无障碍方面提供了组织文化的好处。
在这里，我们提出利用体外原代培养大鼠颈上神经节（SCG）（ 图1）交感神经元模型，研究HSV-1的延迟和重新启动，适合大多数如果不是所有所需的标准。消除非神经细胞后，近同质TrkA的神经元文化与HSV-1感染中存在阿昔洛韦（ACV）的抑制裂解复制。以下阿昔洛韦去除，非生产的HSV-1感染的忠实表现出延迟接受的特点是有效建立。值得注意的是，裂解基因，蛋白质，传染性病毒成为不到，即使在没有选择的情况下，但潜伏相关转录（LAT），快递离子在神经元细胞核坚持。病毒的基因组都保持在25％神经元的平均拷贝数，可以诱导高效复制干扰与PI3激酶/ Akt信号或神经生长因子1的简单撤出。重组HSV-1编码EGFP的融合病毒裂解蛋白Us11提供的复制功能，实时的标记从激活，很容易量化。除了化学处理，如RNA干扰或基因通过慢病毒载体传递基因的方法，可以成功地应用于系统允许是非常困难的，如果不是不可能的，在动物的机理研究。总之，在SCG基于HSV-1的延迟/激活系统提供了一个强大的，必要的工具来解开单纯疱疹病毒潜伏期和活化在神经元，在病毒学的长期难题的解决方案，可提供新的见解，新疗法的开发控制的分子机制目标t他潜伏的疱疹病毒水库。
Protocol
1。从大鼠胚胎SCG神经元的分离和培养 提供有用的背景下，为了解该协议建立的早期文学的全面讨论，上海建工神经元培养方法，包括在体外培养，涂层板，基板，无血清培养基的成分SCG的基础上，读者可以参考参考2-4。 按照NIH的指引下进行的机构动物照顾及使用委员会 （IACUC）批准了一项活动的协议，利用大鼠SCG神经元来源。 前开始清?…
Discussion
这主要神经文化和感染系统提供了一个简单而有效的方法来探讨HSV-1潜伏期和活化的分子机制。该系统忠实地概括公认的标志，在人类的感染和在活的动物模型中定义的延迟。当病毒潜伏在SCG的文化，传染性颗粒和病毒裂解基因产物不能被检测出来。在潜伏感染神经细胞的检测是唯一的病毒基因产物的土地增值税的成绩单，非编码核内积累。激活恰逢病毒裂解基因表达和传染性颗粒生…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢他们周到的建议，有助于改善这个手稿的评论。 MVC（NS21072，HD23315）的ACW（GM61139，S10RR017970）和IM（AI073898，GM056927）从美国国立卫生研究院的赠款，以支持这项工作。旺角是支持部分由美国国立卫生研究院培训资助（5T32 AI007180）。
Materials
| Reagent | Company | Catalog# | Comments |
| 70μm nylon filter( cell strainer) | BD Biosciences | 352350 | |
| 1x Hank’s Balanced Salt Solution (HBSS-/-) | Invitrogen | 14175 | w/o CaCl2 and MgCl2 |
| 1x Minimum Essential Media (MEM) | Invitrogen | 11095-080 | |
| 5-Fluoro-2′-deoxyuridine | Sigma | F0503 | prepare 20 mM stock in 1x MEM; store at -20°C |
| 96-well flat well bottom TC plates | Corning | 3599 | |
| Acyclovir | Calbiochem | 114798 | prepare 31 mM stock in DMSO; store at -20°C |
| Aphidicolin | Calbiochem | 178273 | prepare 10 mM stock in DMSO; store at -20°C |
| B-27 Supplement | Invitrogen | 17504-44 | |
| Collagenase | Sigma | C2674 | prepare 10 mg/ml stock in HBSS-/-; store at -20°C |
| D-(+)-Glucose | Sigma | G6152 | prepare 40% stock in H2O; filter sterilize & store at 4°C |
| L-Glutamine | Invitrogen | 25030-081 | |
| Laminin | Sigma | L2020 | prepare 1 mg/ml stock in H2O; quick-freeze 20 μl aliquats & store at -80°C; dilute to 2 μg/ml working conc. in sterile H2O |
| Leibovit’z L-15 media | Invitrogen | 11415 | |
| Nerve Growth Factor | Harlan Laboratories | BT.5017 | prepare 50 μg/ml stock in HBSS-/-; store at -80°C |
| Neurobasal medium | Invitrogen | 12348 | |
| Phosphonoacetic acid (PAA) | Sigma | P6909 | prepare 75 mg/ml stock in H2O; store at -20°C |
| Poly-D-lysine hydrobromide | Sigma | P0899 | prepare 20 mg/ml stock in H2O; store at -20°C |
| Rat-tail collagen | Millipore | 08-115 | Concentration varies with supply lot; store at 4°C and dilute to 0.66 mg/ml working conc. in sterile H2O |
| Trichostatin A | Sigma | T8552 | prepare 1 mM stock in DMSO; store at -20°C |
| Trypsin 2.5% | Invitrogen | 15090-04 |
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Citar este artigo
Kobayashi, M., Kim, J., Camarena, V., Roehm, P. C., Chao, M. V., Wilson, A. C., Mohr, I. A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation. J. Vis. Exp. (62), e3823, doi:10.3791/3823 (2012).