高糖对高钾刺激引起新生大鼠星形胶质细胞谷氨酸释放和再摄取的影响

目的探讨高糖对高钾刺激引起星形胶质细胞谷氨酸释放和再摄取的影响。方法胶质原纤维酸性蛋白免疫染色法鉴定培养的星形胶质细胞纯度。星形胶质细胞分别用含葡萄糖5,10,20和40 mmol.L-1的细胞外液培养1 h,然后加入氯化钾75 mmol.L-1,于0,1,2,3,4和5 min后分别取细胞培养液。用柱前邻苯二甲醛和N-异丁酰基-D-半胱氨酸衍生高效液相色谱法测定细胞培养液中谷氨酸含量。结果培养的星形胶质细胞纯度在95%以上。不同浓度葡萄糖的细胞外液孵育星形胶质细胞0,1和3 h细胞培养液中谷氨酸含量无明显变化。氯化钾75 mmol.L-1孵育1,2,3,4和5 min后,含葡萄糖10,20和40 mmol.L-1组细胞培养液中谷氨酸含量均比对应时间点含葡萄糖5 mmol.L-1组明显升高(P<0.01),葡萄糖5和10 mmol.L-1组谷氨酸含量在3 min达到峰值,葡萄糖20 mmol.L-1组4 min达峰值;葡萄糖40 mmol.L-1组3 min谷氨酸含量升高最明显,4 min短暂下降后5 min又明显升高。结论高糖能够促进星形胶质细胞谷氨酸的释放和抑制再摄取。

Effect of high glucose on glutamate release and ingestion of astrocytes stimulated by high K + in rats

OBJECTIVE To investigate effect of high glucose on glutamate release and ingestion in rat astrocytes stimulated by high K+.METHODS The cultivated astrocytes were identified by antibody of glial fibrillary acidic protein with immunostaining method.The cultured astrocytes were incubated with glucose 5,10,20 and 40 mmol·L-1 for 1 h,and then stimulated with KCl 75 mmol·L-1 for 0,1,2,3,4 and 5 min.Glutamate concentrations in culture supernatnat at different times were measured with HPLC with precolumn o-phthalaldehyde and N-isobutyryl-D-cysteine derivatization.RESULTS The astrocyte purity was >95%.There was no significant change between glutamate concentrations in culture supernatant with different concentrations of glucose within 3 h.While after stimulation with high KCl,compared of culture supernatant with glucose 5 mmol·L-1,glutamate contents in culture supernatant with glucose 10,20 and 40 mmol·L-1 significantly increased.But changes in glutamate content were different in these groups.In glucose 5 and 10 mmol·L-1 group,the highest content of glutamate in cultured culture supernatant was at 3 min,whereas it was moved to 4 min in glucose 20 mmol·L-1 group,and content of glutamate in culture supernatant had 2 peaks at 3 and 5 min in glucose 40 mmol·L-1 group.CONCLUSIONHigh glucose can affect glutamate content in culture supernatant of astrocytes,which maybe through increasing releasing and inhibiting the uptake of glutamate.