【摘要】 目的:观察银杏内酯B与低氧预适应对氧葡-萄糖剥夺模型中星形胶质细胞的影响,分析该影响作用途径。方法:实验于2004-10/2005-10在南通大学航海医学研究所完成。①选用新生1d的ICR小鼠15只,雌雄不拘。②传3代后的星形胶质细胞,将培养基换成无糖DMEM培养,加入银杏内酯B,置入体积分数0.95空气+体积分数0.05CO2培养箱24h后,进行氧-葡萄糖剥夺实验,培养基换成无糖Hank溶液,置37°C体积分数0.01O2+体积分数0.94N2+体积分数0.05CO2培养箱进行缺氧24h后,换成含150g/L新生牛血清的DMEM/F12完全培养液在体积分数0.95空气+体积分数0.05CO2培养箱复氧24h。③低氧预适应0.5或3h,用原完全培养基培养24h后进行氧-葡萄糖剥夺实验。④利用相差显微镜观察细胞形态变化;四甲基偶氮唑盐比色法测定线粒体脱氢酶活性。免疫印迹分析促红细胞生成素表达的变化。⑤计量资料差异比较采用单因素方差分析,组间差异比较采用Q检验(Newman-Keuls法)。结果:①氧-葡萄糖剥夺实验对星形胶质细胞活性的影响:氧-葡萄糖剥夺0.5或3h后星形胶质细胞活性增强;氧-葡萄糖剥夺24h后,细胞活性明显下降(P<0.01)。②银杏内酯B对氧-葡萄糖剥夺24h后星形胶质细胞活性的影响:预先给予120μmol/L银杏内酯B作用24h,再进行氧-葡萄糖剥夺24h,细胞形态基本正常;在一定浓度范围内银杏内酯B对缺氧损伤有明显的保护作用,以120μmol/L银杏内酯B预处理后细胞活性明显高于氧-葡萄糖剥夺24h后(P<0.01)。③银杏内酯B(120μmol/L)对氧-葡萄糖剥夺24h后星形胶质细胞表达促红细胞生成素的影响:经氧-葡萄糖剥夺0.5或3h或单纯用银杏内酯B处理后的比值明显高于未进行氧-葡萄糖剥夺者(P<0.01);银杏内酯B预处理后再进行氧-葡萄糖剥夺24h表达明显高于氧-葡萄糖剥夺24h后(P<0.05)。④低氧预适应对氧-葡萄糖剥夺24h后星形胶质细胞活性的影响:低氧预适应0.5和3h后再进行氧-葡萄糖剥夺24h,细胞形态基本正常;低氧预适应0.5和3h对氧-葡萄糖剥夺24h后星形胶质细胞有保护作用(P<0.01,0.05)。结论:银杏内酯B与低氧预适应对氧-葡萄糖剥夺星形胶质细胞有保护作用,其作用发生可能与促进促红细胞生成素表达有关。更多还原

【Abstract】 AIM: To observe the effect of Ginkgolide B (Gin B) and hypoxic preconditioning (HP) on astrocytes in the model of oxygen-glucose deprivation (OGD), so as to approach the mechanisms. METHODS: The experiment was conducted in the Institute of Nautical Medicine, Nantong University from October 2004 to October 2005. (1)Fifteen female or male ICR mice 1 day post born were selected. (2) After the medium was changed with DMEM without glucose and added Gin B, the astrocytes subcultured for three times were placed into the incubator for 24 hours containing 0.05 volume fracture CO2, and then OGD experiment was performed. After the medium changed with Hank without glucose, the astrocytes were put in the incubator of 1% O2 + 94% N2 + 5% CO2 for 24 hours for hypoxia, following by in normal incubator of 95% air+5% CO2 with DMEM/F12 complete medium containing 150 g/L fetal bovine serum for 24 hours. (3)HP for 0.5 or 3 hours, and was placed in normal incubator with complete medium for 24 hours for OGD. (4)The morphological changes of astrocytes were observed with phase microscope; dehydrogenase activity of cytochondriome by MTT assay; expression of erythropoietin (EPO) by Western blot analysis. (5)The data were submitted to single factor analysis of variance and the Q-test (Newman-Keuls test) was used to compare data samples among groups. RESULTS: (1)Effect of OGD on astrocytes: The activities of astrocytes enhanced after OGD for 0.5 or 3 hours, and significantly descended after OGD for 24 hours (P < 0.01). (2)Effect of Gin B on activities of astrocytes after OGD for 24 hours: Pretreatment with 120 μmol/L Gin B for 24 hours before OGD 24 hours, the morphology of astrocytes were normal; Gin B at certain concentrations had protective effects on hypoxia injury, the activities of astrocytes after precondition of 120 μmol/L Gin B for 24 hours were obviously higher than that after OGD 24 hours (P < 0.01). (3)Effect of pretreatment with 120 μmol/L Gin B on expression of EPO of astrocytes in OGD 24 hours: Expression of EPO with treatment of 120 μmol/L Gin B or OGD for 0.5 or 3 hours was higher than that without OGD (P < 0.01); and that OGD 24 hours after pretreatment with 120 μmol/L Gin B was higher than OGD 24 hours only (P < 0.05). (4)Effect of HP on the activities of astrocytes in OGD 24 hours: Pretreatment with HP for 0.5 or 3 hours before OGD 24 hours, the morphology of astrocytes were normal; HP had significant protective effects on astrocytes after OGD 24 hours (P < 0.01, 0.05). CONCLUSION: Pretreatment of Gin B and HP have protective effects on astrocytes after OGD 24 hours, which is related to the improvement of expression of EPO.更多还原