体外培养星形胶质细胞缺氧/复氧后EAAT2、谷氨酰胺合成酶的表达

Study of hypoxia/reoxygenation on expression of excitatory amino acid transporter-2 and glutamine synthetase in cultured astrocytes

目的对体外培养的星形胶质细胞（AC）进行缺氧／复氧及亚低温干预。观察兴奋性氨基酸转运体2（EAAT2）、谷氨酰胺合成酶（GS）的表达及亚低温干预对其的影响。方法选用新生大鼠的大脑皮层组织培养的AC。分为对照组（C）、缺氧／复氧组（H／R）、亚低温组（H／M＋R），缺氧12h后H／R组及H／M＋R组分别于37℃及32℃复氧。每组设复氧2h、4h、8h、12h、24h、48h等时间点。MTT测定细胞活力，Western—blot半定量分析EAAT2、GS的表达情况。结果①MTT法检测细胞活力：H／R组与H／M＋R组的细胞活力均比C组的细胞活力低，而H／M＋R组的细胞活力较H／R组高，差异有统计学意义（P〈0．05）。（2）Western—blot检测结果：H／R组与H／M＋R组的EAAT2表达与C组相比较，均先减少，后随着复氧时间延长逐渐增多，差异有统计学意义（P〈0．05），H／M＋R组的EAAT2表达与H／R组相比较，表达量下降趋势小，差异有统计学意义（P〈0．05）；H／R组与H／M＋R组的GS表达量随着复氧时间延长。均呈现增高趋势，与C组比较增高显著，差异有统计学意义（P〈0．05），H／M＋R组的GS表达量与H／R组比较增高趋势更加明显，差异有统计学意义（P〈0．05）。结论缺氧／复氧及亚低温干预可引起星形胶质细胞EAAT2、GS表达的改变：32℃～35℃亚低温干预可以抑制缺氧／复氧损伤后兴奋性氨基酸的释放，这可能是脑保护作用的重要机制之一。

Objective To observe the expression of excitatory amino acid transporter-2 （EAAT2） and glutamine syntbetase （GS） along with the effect of mild hypothermia on them in cultured astrocytes in vitro after hypoxia/reoxygenation and mild hypothermia. Methods Cultured corticocerebral astrocytes （ACs） in neonatal rats were divided into three groups： control group （C）, hypoxia/reoxygenation group （H/R） and mild bypothermia plus reoxygenation group （H/M＋R）. H/R and H/M＋R group were reoxygenated at 37℃ and 32℃ for 12 hours after hypoxia, respectively. Each group was reoxygenated at following time points ：2b, 4h, 8h, 12h, 24h and 48h. Cell viability was assayed by MTT. Semi-quantitative analysis was made on the expression of EAAT2 and GS by Western-blot. Results （1）Cell viability by MTT assay ：The cell viability in either H/R or H/M＋ R group was weaker than that in the control group, while that in the H/M＋R group was stronger than that in the H/R group;the difference was statistically significant（P〈0.05）. （2）Western-blot findings：Compared with the control, expression of EAAT2 firstly decreased and then increased over reoxygenation time in either H/R or H/M＋R group; expression of EAAT2 was lower in the H/M＋R group compared with the H/R group;expression of GS significantly increased over reoxygenation time in either H/R or H/M＋R group compared with the control;expression of GS was significantly higher in the H/M＋R group than that in the H/R group. All these differences were statistically significant （P〈0.05）. Conclusion Both hypoxia/reoxygenation and mild hypothermia can lead the changes in expression of EAAT2 and GS in astrocytes; mild hypothermia at 32℃ to 35℃ can suppress the release of excitatory amino acids after hypoxia/reoxygenation damage, indicative of a possible important mechanism of cerebral protection.