Methodological limitations in determining astrocytic gene expression.
Methodological limitations in determining astrocytic gene expression.
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DOI：
10.3389/fendo.2013.00176
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发表时间：
2013-11-25
影响因子：
5.2
通讯作者：
Wang Z
中科院分区：
文献类型：
--
作者：
Peng L;Guo C;Wang T;Li B;Gu L;Wang Z
传统上，星形细胞 mRNA 和蛋白质表达通过原位杂交 (ISH) 和免疫组织化学进行研究。这导致了星形胶质细胞缺乏aralar的概念，aralar是苹果酸-天冬氨酸-穿梭的一个组成部分。至少通过荧光辅助细胞分选 (FACS) 分离的星形胶质细胞和神经元中类似的 aralar mRNA 和蛋白质表达推翻了这一观点。其他星形细胞基因表达的证明也可能是错误的。因此，将基于形态学方法的文献数据与通过最近开发的细胞特异性基因表达测定方法获得的细胞中的mRNA表达进行比较。所有 Na,K-ATPase-α 亚基均通过免疫组织化学 (IHC) 进行了证明，但协同转运蛋白 NKCC1 存在问题。免疫组织化学可以很好地测定谷氨酸和 GABA 转运蛋白基因的表达。这同样适用于许多葡萄糖代谢基因的表达，而基于细菌人工染色体（BAC）转基因动物的一项研究显示，己糖激酶的星形胶质细胞表达非常低。 ISH 识别平衡核苷转运蛋白 ENT1 和 ENT2 的基因表达，但 ENT3 不能。这同样适用于集中转运蛋白 CNT2 和 CNT3。所有这些都在 FACS 分离的细胞中清晰表达，然后进行生化分析。 ENT3 在星形胶质细胞中富集。微阵列分析显示了许多核苷转运蛋白基因的表达，而其他重要基因则没有。培养的星形胶质细胞的结果与 FACS 获得的结果相似。这些发现需要重新评估细胞核苷转运蛋白的表达。 FACS 细胞产量较小。进一步开发细胞分离方法，使方法更容易获得，减少动物和成本消耗，并通过 ISH/IHC 和其他方法对星形胶质细胞 mRNA 和蛋白质表达进行平行研究是必要的，但新方法也需要彻底检查。
Traditionally, astrocytic mRNA and protein expression are studied by in situ hybridization (ISH) and immunohistochemically. This led to the concept that astrocytes lack aralar, a component of the malate-aspartate-shuttle. At least similar aralar mRNA and protein expression in astrocytes and neurons isolated by fluorescence-assisted cell sorting (FACS) reversed this opinion. Demonstration of expression of other astrocytic genes may also be erroneous. Literature data based on morphological methods were therefore compared with mRNA expression in cells obtained by recently developed methods for determination of cell-specific gene expression. All Na,K-ATPase-α subunits were demonstrated by immunohistochemistry (IHC), but there are problems with the cotransporter NKCC1. Glutamate and GABA transporter gene expression was well determined immunohistochemically. The same applies to expression of many genes of glucose metabolism, whereas a single study based on findings in bacterial artificial chromosome (BAC) transgenic animals showed very low astrocytic expression of hexokinase. Gene expression of the equilibrative nucleoside transporters ENT1 and ENT2 was recognized by ISH, but ENT3 was not. The same applies to the concentrative transporters CNT2 and CNT3. All were clearly expressed in FACS-isolated cells, followed by biochemical analysis. ENT3 was enriched in astrocytes. Expression of many nucleoside transporter genes were shown by microarray analysis, whereas other important genes were not. Results in cultured astrocytes resembled those obtained by FACS. These findings call for reappraisal of cellular nucleoside transporter expression. FACS cell yield is small. Further development of cell separation methods to render methods more easily available and less animal and cost consuming and parallel studies of astrocytic mRNA and protein expression by ISH/IHC and other methods are necessary, but new methods also need to be thoroughly checked.