长期传代诱导的细胞衰老会对 C6 细胞系中的谷氨酸摄取产生不利影响。

Cellular senescence induced by prolonged subculture adversely affects glutamate uptake in C6 lineage.

机构信息

Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), Rua Ramiro Barcelos, 2600 prédio anexo, Porto Alegre, RS, 90035-003, Brazil,

出版信息

Neurochem Res. 2014 May;39(5):973-84. doi: 10.1007/s11064-014-1295-1. Epub 2014 Apr 5.

DOI:10.1007/s11064-014-1295-1

PMID:24706093

Abstract

Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60-180 min of L-[(3)H]glu incubation, LPC6 presented an intracellular [(3)H] 55% lower than EPC6. Both cultures showed a time-dependent increase of intracellular [(3)H] reaching maximal levels at 120 min. Cultures incubated with D-[(3)H]asp showed a time-dependent increase of [(3)H] until 180 min. Moreover, LPC6 have a D-[(3)H]asp-derived intracellular [(3)H] 30-45% lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50% of L-[(3)H]glu-derived intracellular [(3)H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high β-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters.

摘要

最近，几位研究人员使用 C6 细胞来评估高亲和力谷氨酸转运体的功能特性。然而，已经证明，根据培养传代数的不同，该谱系会发生几种形态和生化改变。目前，尚无报道表明高亲和力谷氨酸转运体的功能特性是否符合这些亚培养依赖性的改变。本研究旨在通过详细的药理学和生化特征比较早期（EPC6）和晚期（LPC6）传代 C6 细胞中表达的高亲和力谷氨酸转运体的功能特性。在 L-[(3)H]Glu 孵育 60-180min 期间，LPC6 的细胞内[(3)H]比 EPC6 低 55%。两种培养物均表现出时间依赖性的细胞内[(3)H]增加，在 120min 时达到最大水平。用 D-[(3)H]asp 孵育的培养物在 180min 内也表现出时间依赖性的[(3)H]增加。此外，LPC6 的 D-[(3)H]asp 衍生的细胞内[(3)H]在 120min 之前比 EPC6 低 30-45%。只有 EAAT3 在培养物中被免疫检测到，并且它们的总含量相等。PMA 刺激 EAAT3 向膜转运增加了 EPC6 中 L-[(3)H]Glu 衍生的细胞内[(3)H]的 50%，而对 LPC6 没有影响。LPC6 表现出类似于衰老的特征，例如高β-Gal 染色、细胞增大和大而规则核的增加。我们的结果表明，LPC6 表现出谷氨酸摄取受损，这可能是由于其无法将 EAAT3 动员到细胞膜所致。这种表型可能与该培养物中观察到的衰老过程有关。我们的结果表明，LPC6 细胞是研究高亲和力谷氨酸转运体功能特性的不合适的神经胶质细胞模型。