Vergé V, Legrand A, Hévor T
Laboratoire de Physiologie, CNRS-UMR 1294, Université d'Orléans, France.
Glia. 1996 Nov;18(3):244-54. doi: 10.1002/(SICI)1098-1136(199611)18:3<244::AID-GLIA8>3.0.CO;2-Z.
Astrocytes are the principal sites of glycogen synthesis in the nervous tissue. Growing evidence shows that there are many types of astrocytes. The aim of the present investigation was to isolate different types of astrocytes that display different carbohydrate anabolism. Astrocytes from newborn rat brain were directly cloned from primary cultures without a previous transformation. Many clones were obtained, and they were termed CP clones. Another series of clones, termed SV clones, were obtained after the transfection of the primary cultures by the SV40 T antigen. The effectiveness of the transfection was verified by the rate of DNA synthesis using flow cytometry and by the presence of plasmid DNA in the genomic DNA of the astrocytes using the Southern blot method. After the transfection, the growth velocity increased greatly. The size and shape of the astrocytes were the same for each cell in a given clone, regardless of the cloning method utilized. However, these sizes and shapes could be different from one clone to another in CP clones, whereas all the astrocytes of all the SV clones looked like each other. All the clones obtained stained positively with anti-glial fibrillary acidic protein antibodies. Glycogen stained in the clones using concanavalin A-horseradish peroxidase. The glycogen content was also measured using biochemical analysis. Concordant results obtained using two methods showed that some clones contained an important quantity of glycogen while other clones contained a small amount, in the CP series as well as in the SV series. This property was the same for the intracellular glucose concentrations. The activity of the gluconeogenic enzyme fructose-1, 6-bisphosphatase was measured in each clone using spectrophotometry. This activity was also significantly different from one clone to another. The clones containing large amounts of glycogen had important fructose-1,6-bisphosphatase activity. The present results show that it is possible to clone astrocytes either directly from primary cultures without immortalization or after their transformation. When analyzing these clones, it appears that carbohydrate anabolism can be significantly different from one astrocyte to another. This difference may also exist in vivo.
星形胶质细胞是神经组织中糖原合成的主要场所。越来越多的证据表明存在多种类型的星形胶质细胞。本研究的目的是分离出表现出不同碳水化合物合成代谢的不同类型星形胶质细胞。新生大鼠脑内的星形胶质细胞直接从原代培养物中克隆得到，未经预先转化。获得了许多克隆，它们被称为CP克隆。另一系列克隆，称为SV克隆，是在原代培养物用SV40 T抗原转染后获得的。通过流式细胞术检测DNA合成速率以及用Southern印迹法检测星形胶质细胞基因组DNA中质粒DNA的存在来验证转染的有效性。转染后，生长速度大大提高。对于给定克隆中的每个细胞，无论采用何种克隆方法，星形胶质细胞的大小和形状都是相同的。然而，在CP克隆中，这些大小和形状在不同克隆之间可能不同，而所有SV克隆的星形胶质细胞彼此看起来相似。所有获得的克隆用抗胶质纤维酸性蛋白抗体染色均呈阳性。用伴刀豆球蛋白A - 辣根过氧化物酶对克隆中的糖原进行染色。还用生化分析测量了糖原含量。两种方法获得的一致结果表明，在CP系列以及SV系列中，一些克隆含有大量糖原，而其他克隆含有少量糖原。细胞内葡萄糖浓度也有相同情况。用分光光度法在每个克隆中测量糖异生酶果糖 - 1,6 - 二磷酸酶的活性。这种活性在不同克隆之间也有显著差异。含有大量糖原的克隆具有重要的果糖 - 1,6 - 二磷酸酶活性。目前的结果表明，既可以直接从原代培养物中克隆星形胶质细胞而不使其永生化，也可以在其转化后进行克隆。在分析这些克隆时，似乎碳水化合物合成代谢在不同星形胶质细胞之间可能有显著差异。这种差异在体内也可能存在。
