雪旺的星形胶质细胞使用相互作用分析<em在体外检测
Summary
本文拟在逐步时尚来形容常用的<em>在体外</em> asrtocyte雪旺氏细胞相互作用研究中使用的检测。
Abstract
雪旺氏细胞修复策略常用的脊髓损伤后的细胞之一。雪旺氏细胞能够支持轴突再生和分泌生长因子1,2的发芽和提供增长，促进粘附分子和细胞外基质分子4。此外，他们还myelinate脱髓鞘轴突损伤5现场。
但移植后，雪旺氏细胞不从植入部位迁移，不混杂与主机的星形胶质细胞6,7。这样的结果形成的雪旺氏细胞和星形胶质细胞之间的尖锐边界，试图退出到宿主组织移植回近端和远端轴突增长的一个障碍。星形胶质细胞与雪旺氏细胞接触也经历肥大，上调8-13抑制分子。
在体外实验中已被用于模型雪旺氏细胞，星形胶质细胞相互作用，并已在了解细胞行为的机制的重要。
在体外试验 ，其中包括边界检测，其中一个共同的文化占领只是一个很小的分离两种细胞方面的差距与不同地区的每一个细胞类型中使用两种不同的细胞。随着细胞分裂和迁移，两个细胞战线得到彼此更加接近并最终相撞。这使得边界分析的两个细胞群的行为。同样的技术的另一个变化是混合文化中的两个细胞群，并随着时间的推移，两种类型的细胞与雪旺氏细胞分隔成群一起共同创建多个雪旺星形胶质细胞的界限的星形胶质细胞之间的岛屿。
第二个实验研究两种类型的细胞的相互作用是可以对其他类型的细胞单层 14,15表面跟踪的细胞运动迁移实验。这个实验是俗称倒盖玻片检测。雪旺氏细胞培养小玻璃碎片和面倒立到星形胶质细胞单分子膜的表面和迁移是从盖玻片边缘的评估。
两种检测已在研究参与细胞的排斥和边界形成的基本机制。一些分子确定使用这些技术包括N -钙粘素，硫酸软骨素蛋白多糖（CSPGs）16,17，FGF /肝素18，弗/ Ephrins 19。
本文拟来形容边界检测，并在逐步时尚迁移实验和澄清可能出现的技术可能出现的问题。
Protocol
1。边界含量： 基本准备：玻片过夜，无菌的D -聚赖氨酸的载玻片涂在开始实验前准备。中等准备使用贝科改良Eagle培养基（DMEM），辅以10％小牛血清（FCS），和1％的青霉素，链霉素Fungizon（PSF）。另外，准备一瓶免费汉克斯钙/镁平衡盐溶液（HBSS）。 烧瓶中培养的原代大鼠雪旺细胞胰酶消化3分钟，用0.1％胰蛋白酶。 5分钟，用0.1％胰蛋白酶原代大鼠星形胶质细胞?…
Discussion
上文所述的检测已用于各种研究证明在边界之间的雪旺星形胶质细胞和星形胶质细胞的环境有限的雪旺细胞迁移的形成涉及多种因素的作用。
了解这些事件的内在机制是至关重要的，因为这将使发展战略，优化和提高移植后的雪旺氏细胞移植的一体化，并在这样做方便出口到主机，让组织移植回再生轴突形成连接与宿主组织。
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢她的帮助菲莉帕沃伦。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| DMEM | Invitrogen | 41966-029 | ||
| HBSS | Invitrogen | 14170-088 | ||
| FCS | Invitrogen | 10091-148 | ||
| PSF | Invitrogen | 15240-062 | ||
| BPE | Invitrogen | 13028-014 | ||
| Forskolin | Calbiochem | 344273 | ||
| Coverlips | VWR-International | 631-0149 | ||
| Coverlips(rectangle) | MENZEL-GLASER (Supplied by Thermo Scientifics) | BB022050A1 | ||
| Chamber slides | Nunc-LabTek | 177380 | ||
| 4 well plates, 6 well plates | Nunc | 4 well plates 6 well plates |
||
| rat p75 antibody | Millipore, Temecula,California | MAB365 | ||
| GFAP antibody | Dako | Z0334 | ||
| Secondary antibodies (Alexa-conjugated) | Invitrogen | A-11004 A-11034 |
Goat anti mouse 568 Goat anti Rabbit 488 |
|
| Secondary biotinylated | Vector | Goat anti mouse | ||
| DAB tablets | Sigma-aldrich | D4418 | ||
| ABC elite kit | Vector | PK-6100 | ||
| Fine Forceps | FST | 11295-10 | ||
| Paraformaldehyde | Sigma-Aldrich | P6168 | ||
| Fluorosave | Calbiochem | 345789 |
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Cite This Article
T. Afshari, F., C. Kwok, J., W. Fawcett, J. Analysis of Schwann-astrocyte Interactions Using In Vitro Assays. J. Vis. Exp. (47), e2214, doi:10.3791/2214 (2011).