氧气葡萄糖剥夺诱导原代培养的星形胶质细胞释放谷氨酸

Oxygen glucose deprivation induced glutamate release in primary cultured astrocytes

目的观察氧气葡萄糖剥夺(OGD)对原代培养的星形胶质细胞谷氨酸释放的影响,并探讨其释放机制。方法原代培养SD大鼠海马区星形胶质细胞,将其分为OGD组和对照组。OGD组的细胞置于不含糖和氧的培养基,37℃,950 mL/L N2和50 mL/L CO2,饱和湿度的培养环境下培养,而对照组细胞则正常培养。缺糖缺氧刺激时长分别为0、15、30、60、90、120 min,采用高效液相色谱,测定细胞外液的谷氨酸浓度。分别选用连接子蛋白43(Cx43)特异性反义寡核苷酸(Cx43-ASODN)和Cx43半通道的阻断剂Gap26预处理星形胶质细胞,采用高效液相色谱,测定细胞外液谷氨酸浓度,观察OGD对其谷氨酸释放的影响。结果与对照组相比较,OGD刺激后,细胞外液谷氨酸浓度升高,并在刺激90 min后,达到峰值,为(5.00±0.30)nmol/mL,显著高于对照组的(2.36±0.15)nmol/mL(P<0.05);而OGD条件下,Cx43-ASODN或Cx43半通道阻断剂均可抑制细胞外液谷氨酸浓度的升高,刺激90 min后,细胞外液谷氨酸浓度分别为(4.02±0.18)nmol/mL和(3.93±0.32)nmol/mL,显著低于单纯OGD刺激组(P<0.05)。结论 OGD可以诱导星形胶质细胞通过Cx43半通道释放谷氨酸。

Objective To investigate the effect of oxygen glucose deprivation （OGD） on the glutamate release and the role of connaxin 43 （ Cx43 ） hemichannels in the OGD-induced glutamate release in primary cultured astrocytes. Methods The astrocytes were randomly divided into four groups： normal control, OGD group, Gap26 （Cx43 hemichannel blocker） plus OGD group, Cx43-specific antisense oligodeoxynucleoUde （ASODN） p!us OGD group. In the OGD group, the astrocytes were cultured in the oxygen- and glucose-free culture medium （950 mL/L N= and 50 mL/L CO2 ） for different durations （0, 15, 30, 60, 90, 120 minutes） at 3723, while the astrocytes in the control group were cultured in the ordinary culture medium. In the Gap26 plus OGD group or Cx43-ASODN plus OGD group, the astrocytes were incubated with Gap26 or Cx43-ASODN before and dudng the OGD. The extracelluar glutamate level was measured by high performance liquid chromatography （HPLC）. Results After OGD, the extracelluar glutamte level significantly increased, and reached the maximum （5.00 ± 0.30 ） nmol/mL at 90 minutes of OGD, which was significantly higher than that of the normal control （2.36 ±0. 15） nmoVmL（P〈0.05）. After treatment by Cx43-ASODN or Gap26, the increase was inhibited. At 90 minutes of OGD, the extracellular glutamate levels were respectively （4.02 ±0.18） nmol/mL and （3.93 ±0.32） nmol/mL. They were signficalty lower than that of the OGD group at the same time （ P 〈 0.05）. Conclusion OGD induced glutamate release through Cx43 hemichannel in primary cultured astrocytes.