Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-L-lactic acid fibers.
Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-L-lactic acid fibers.
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DOI：
10.1016/j.biomaterials.2013.10.079
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发表时间：
2014-02
期刊：
影响因子：
14
通讯作者：
Gilbert, Ryan J.
中科院分区：
文献类型：
--
作者：
Zuidema, Jonathan M.;Hyzinski-Garcia, Maria C.;Van Vlasselaer, Kristien;Zaccor, Nicholas W.;Plopper, George E.;Mongin, Alexander A.;Gilbert, Ryan J.
关键词：
生物工程纤维基质作为一种促进受损中枢神经系统（CNS）再生和重塑的手段，越来越多地被研究。以前的报道主要集中在定向支架桥接损伤区域和轴突投射直接生长的能力上。在本工作中，我们探讨了静电纺丝微纤维对脑星形胶质细胞的迁移和生理特性的影响。原代大鼠星形胶质细胞在纤连蛋白涂覆的聚-L-乳酸（PLLA）膜、纤连蛋白涂覆的随机取向的PLLA电纺纤维或纤连蛋白涂覆的对齐的PLLA电纺纤维上培养。对齐PLLA纤维强烈改变星形胶质细胞形态，定向细胞过程，肌动蛋白微丝，和微管沿着纤维的长度。在对齐的纤维上，星形胶质细胞也显著增加了它们在纤维取向方向上的迁移率。我们进一步研究了纤维拓扑结构是否改变了星形胶质细胞的神经保护特性，即谷氨酸和谷氨酰胺的转运和代谢。这是通过定量一组相关生物分子的mRNA表达（qRT-PCR）和蛋白质水平（Western印迹）的变化来完成的。有趣的是，我们发现，与纤连蛋白包被的膜相比，在随机和/或对齐的纤维上生长的细胞增加了两种谷氨酸转运蛋白GLAST和GLT-1以及一种重要的代谢酶谷氨酰胺合成酶的表达水平。功能测定显示，由于GLT-1介导的摄取，谷氨酸转运速率增加，这在很大程度上是由二氢红藻氨酸敏感的GLT-1。总体而言，这项研究表明，对齐PLLA纤维可以促进定向星形胶质细胞迁移，最重要的是，我们的体外研究结果表明，第一次电纺PLLA纤维可以积极修改神经胶质细胞的神经保护性能，通过增加谷氨酸摄取率。
Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-l-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake.
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作者：
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DOI：
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