Peng Liang, Guo Chuang, Wang Tao, Li Baoman, Gu Li, Wang Zhanyou
Department of Clinical Pharmacology, China Medical University , Shenyang , China.
Front Endocrinol (Lausanne). 2013 Nov 25;4:176. doi: 10.3389/fendo.2013.00176.
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.
传统上，星形胶质细胞的mRNA和蛋白质表达是通过原位杂交（ISH）和免疫组织化学方法进行研究的。这导致了星形胶质细胞缺乏苹果酸 - 天冬氨酸穿梭体的一个组成部分——丙氨酸载体（aralar）的概念。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至少相似的丙氨酸载体mRNA和蛋白质表达改变了这一观点。其他星形胶质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用于确定细胞特异性基因表达的方法获得的细胞中的mRNA表达进行了比较。所有Na，K - ATP酶-α亚基都通过免疫组织化学（IHC）得到证实，但协同转运蛋白NKCC1存在问题。谷氨酸和GABA转运体基因表达通过免疫组织化学得到很好的确定。许多葡萄糖代谢基因的表达也是如此，而基于细菌人工染色体（BAC）转基因动物研究结果的一项单独研究表明己糖激酶的星形胶质细胞表达非常低。平衡核苷转运体ENT1和ENT2的基因表达通过ISH得到确认，但ENT3未得到确认。集中转运体CNT2和CNT3也是如此。所有这些在FACS分离的细胞中均有明显表达，随后进行生化分析。ENT3在星形胶质细胞中富集。通过微阵列分析显示了许多核苷转运体基因的表达，而其他重要基因则未显示。培养的星形胶质细胞中的结果与通过FACS获得的结果相似。这些发现需要重新评估细胞核苷转运体的表达。FACS获得的细胞产量很少。需要进一步开发细胞分离方法，使方法更易于获得，减少动物使用和成本消耗，并通过ISH/IHC和其他方法对星形胶质细胞的mRNA和蛋白质表达进行平行研究，但新方法也需要进行彻底检查。
