Localized gene expression of axon guidance molecules in neuronal co-cultures
Localized gene expression of axon guidance molecules in neuronal co-cultures
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DOI：
10.1016/j.jneumeth.2006.07.013
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发表时间：
2007-01-30
影响因子：
3
通讯作者：
Snow, D. M.
中科院分区：
文献类型：
--
作者：
Heron, P. M.;Sutton, B. M.;Snow, D. M.
轴突生长锥通过接触依赖性机制或扩散性趋化因子导向其靶点。通过这些因子的轴突引导通常涉及在可能不代表体内生物环境的无细胞基质上培养神经元。我们开发了两种新的体外方法，以在生理相关的细胞环境中创建引导分子的模式化基因表达。在Matrigel（TM）测定中，将腺病毒-Matrigel（TM）悬浮液的液滴置于生长在Matrigel（TM）中的星形胶质细胞上。腺病毒通过凝胶扩散并转导下面的星形胶质细胞，在局部区域内产生径向感染梯度。在第二个模型中，重组腺病毒与吸附在硝酸纤维素条纹图案上的抗六邻体抗体结合。一旦加入细胞，只有那些接触腺病毒的细胞被感染。本文研究了鸡背根神经节神经元在神经生长因子（NGF）、脑信号蛋白3A（semaphorin 3A）和短蛋白聚糖（brevican）作用下的生长模式。正如预期的那样，结果显示了与分泌的Sema 3A或膜结合的短蛋白聚糖相反的朝向NGF的稳健轴突生长，然而，与用较少生理相关的方法获得的那些相比，观察到轴突生长反应的细微差异。这种技术的新颖之处在于，可以在复杂的细胞环境中控制和操纵分子表达的位置和区域。(c)2006 Elsevier B. V.保留所有权利。
Axonal growth cones are guided to their targets by contact-dependent mechanisms or by diffusible chemotropic factors. Axon guidance by these factors typically involves culturing neurons on an acellular substrate which may not represent the in vivo biological environment. We developed two novel in vitro methods to create patterned gene expression of guidance molecules in a physiologically-relevant cellular environment. In the Matrigel (TM) assay, a droplet of adenovirus-Matrigel (TM) suspension was placed on astrocytes grown in Matrigel (TM). The adenovirus diffused through the gel and transduced underlying astrocytes, creating a radial infection gradient within a localized area. In the second model, recombinant adenovirus was bound to an anti-hexon antibody adsorbed onto stripe patterns of nitrocellulose. Once the cells were added, only those contacting the adenovirus were infected. The outgrowth pattern of chick DRG neurons on NGF, semaphorin 3A and brevican were studied. As expected, results showed robust axonal growth toward NGF as opposed to either secreted Sema 3A or membrane bound brevican, however subtle differences in axonal growth responses were observed in comparison to those obtained with less physiologically-relevant methods. Novel to this technology, the location and area of molecule expression can be controlled and manipulated in an intricate cellular environment. (c) 2006 Elsevier B.V. All rights reserved.