缺氧复氧时体外培养的神经细胞一氧化氮合酶的动态表达及银杏叶提取物的调节作用

本文研究了缺氧复氧时原代培养的大鼠神经细胞 NOS的动态表达及银杏叶提取物的调节作用 ,藉以探讨 NO在缺血缺氧导致神经细胞损伤时的作用以及银杏叶提取物的保护机制。实验使用胎龄 15～ 17d大鼠的大脑皮层神经细胞进行原代分离培养 ,建立缺氧复氧神经元损伤模型。实验分为正常对照组、单纯缺氧组、缺氧复氧组和银杏叶提取物处理组。应用 NADPH-d组织化学方法观察神经细胞 NOS的动态表达 ,并用图象分析仪进行定量检测。结果 :缺氧复氧可以使神经细胞的 NOS呈双时相的升高 :在单纯缺氧 2、4、6、8h组及缺氧加复氧 18h组中 ,缺氧 2 h组及缺氧 8h复氧 18h组与对照组相比 NOS阳性细胞数量增多 ,染色加深 ,酶活性增强 ,经统计差异具有显著性 (P<0 .0 1)。其它组与对照组比较无明显差异。中药银杏叶提取物可以抑制上述两组的NOS活性增强。本文结果提示 :缺氧及缺氧复氧可以使神经细胞 NOS活性呈双时相增强 ,NO的升高可能是缺氧复氧导致神经细胞损伤的原因之一 ;银杏叶提取物对缺氧复氧导致的神经细胞损伤的保护作用可能是通过下调 NOS活性而实现的

EFFECTS OF EGB ON THE DYNAMIC EXPRESSION OF NOS IN PRIMARY CULTURED NEURONS IN RATS FOLLOWING HYPOXIA/REOXYGENATION

The study is to investigate the dynamic expression of nitric oxide synthase(NOS) in primary cultured rat cortical neurons following hypoxia reoxygenation(H/R) and the effects of extract of ginkgo biloba(EGB) on the expression of NOS in order to explore the role of NO in H/R induced neuronal damage and the mechanisms of EGB's neuronal protection. Primary culture of E16～17 days fetal rat cortical neurons was used to establish H/R injury model. There were 4 groups : (1)normal control group, (2)hypoxia only group(H), (3)hypoxia/reoxygenation group(H/R), (4)EGB group(EGB). For each group, the expression of NOS was examined by NADPH diaphorase histochemical staining and the quantitative changes of NOS were determined by image analysis system (Leica). Results: (1)The number of NOS positive cell increased dramatically in 2 hour hypoxia group(H 2R 0) compared to that in the normal control group, and the staining of NOS was stronger than that in the normal control group. In accordance with the histochemical observation, quantitative analysis showed significant increase of NOS activity (P<0.01) in H 2R 0 group compared to the normal control group as well as the 4 h, 6 h, 8 h hypoxia groups (H 4R 0, H 6R 0, H 8R 0). In H/R group, the expression of NOS was increased in 8 h hypoxia 18 h reoxygenation group(H 8R 18 )(P<0.05) compared to that in the normal control group. However, there was no statistically significant change in H 2R 18 , H 4R 18 , H 6R 18 groups compared to the normal control group. (2)The increase of NOS activity was effectively blocked by pre incubation with EGB(50 μg/ml) 1 h before hypoxia in H 2R 0 and H 8R 18 groups. Conclusion: There was a bi phasic increase of NOS expression during hypoxia(H 2R 0) and hypoxia/reoxygenation(H 8R 18 ). This could be one of the reasons resulting in neuronal damage. The protective effect of EGB on neuronal damage could be due to its inhibition of the activity of NOS, hence, reducing the production of NO in this model.