解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染
Summary
mechanosensory毛细胞是内耳的受体细胞。最好的特点<em>在体外</em成熟的哺乳动物毛细胞模型系统利用从成年鼠的椭圆囊器官培养。我们目前的成年小鼠的囊的剥离，我们证明腺病毒介导的支持细胞在体外培养椭圆囊的感染。
Abstract
听力损失和平衡紊乱往往造成mechanosensory毛细胞是内耳的受体细胞死亡。由于没有细胞行圆满代表了哺乳动物的毛细胞，毛细胞的研究依赖于基层机关文化。最好具有成熟的哺乳动物毛细胞在体外模型系统，利用从成年鼠（ 图1）1-6椭圆囊器官培养。囊是前庭器官，以及椭圆囊毛细胞的听觉器官，Corti器毛细胞结构和功能相似。成年小鼠的囊准备成熟的感觉上皮细胞的生存，动态平衡，这些细胞死亡的分子信号调节的研究。
哺乳动物耳蜗毛细胞的终末分化，当他们失去了，不能再生。在非-MAM其次是强大的再生，恢复听力和平衡功能7，8，马里脊椎动物，听觉或前庭毛细胞死亡。再生毛细胞介导的胶质状的支持细胞，毛细胞的基底表面的接触中感觉上皮9，10。支持细胞是重要的调停毛细胞生存和死亡的11。我们最近开发出一种技术，支持在使用培养椭圆囊腺病毒的细胞感染。利用腺病毒5型（dE1/E3）提供含有CMV启动子控制下的绿色荧光蛋白的转基因，我们发现，腺病毒特别有效地支持细胞的感染。支持细胞的感染效率约为25-50％，并没有感染毛细胞（ 图2）。重要的是，我们发现，不支持细胞的腺病毒感染导致毛细胞或支持细胞的毒性，因为在AD-GFP INF细胞计数ected椭圆囊相当于在非疫区的椭圆囊（ 图3）。因此，在支持细胞培养椭圆囊腺病毒介导的基因表达提供了一个功能强大的工具，研究支持细胞作为调解人毛细胞生存，死亡和再生的作用。
Protocol
Discussion
感觉毛细胞受到的各种压力，包括老龄化，噪声外伤，和接触，包括氨基糖甙类抗生素和抗癌药顺铂的耳毒性药物引起的死亡。在哺乳动物毛细胞，导致永久性听力丧失和/或平衡紊乱导致死亡。 在体外模型系统研究旨在确定的细胞和分子机制的毛细胞死亡，以及那些旨在防止或扭转毛细胞死亡的关键工具。不同于成年哺乳动物耳蜗毛细胞，毛细胞的囊良好的生存文化。囊毛细胞是敏感的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家耳聋与其他交流障碍研究所院内研究司。 NIDCD的5R01 DC007613提供了额外的支持。
Materials
| Reagent | Company | Catalog number |
| Medium 199 | Gibco/Invitrogen | 12350 |
| DMEM/F12 | Gibco/Invitrogen | 11320 |
| Fine forceps (#55) | Fine Science Tools | 11255-20 |
| Fine forceps (#5) | Fine Science Tools | 11252-30 |
| Forceps (#3) | Fine Science Tools | 11231-30 |
| Penicillin G | Sigma | P3032 |
| Fetal bovine serum | Invitrogen | 10082-139 |
| Nunc mini-tray | Fisher | 12-565-68 |
| Adenovirus-GFP | Vector Biolabs | 1060* |
| Cal-Ex decalcifier | Fisher | CS510-1D |
| sodium borohydride | Sigma | 452882 |
| Anti-Myosin 7a (polyclonal) | Proteus Biosciences | 25-6790 |
| Anti-Myosin 7a (monoclonal) | Developmental Studies Hybridoma Bank | MYO7A 138-1 |
| Anti-Calmodulin | Sigma | C 3545 |
| Anti-Calbindin | Chemicon | AB1778 |
| Fluoromount G | Southern Biotechnology Associates | 0100-01 |
Table 1. Reagents and tools used in utricle culture and adenovirus infection.
* Note: Ad-GFP is normally provided by Vector Biolabs at a stock titer of 1×1010 PFU/ml. We requested a custom amplification in order to provide the stock virus at a titer of 1.2×1011 PFU/ml.
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