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目的:观察亚低温治疗对氧糖剥夺神经细胞凋亡和自噬的影响。方法:提取SPF级SD大鼠新生鼠(1d内)大脑皮层神经元细胞,建立氧糖剥夺/复糖复氧(OGD/R)模型。OGD 3 h后从细胞培养盒中取出细胞培养板,分别置于37℃或34℃恒温培养箱中复氧6 h、12 h。western-blot检测凋亡蛋白P53蛋白和自噬相关蛋白LC3Ⅱ/Ⅰ、Beclin1、Atg5和Rab7的表达。Tunel法检测神经元细胞凋亡。结果:新生鼠原代神经元分离、培养、接种4h后贴壁良好。常温组,原代神经元OGD3h,复氧后6h和12h,western-blot Rab7表达逐渐减少;Beclin1表达减少;LC3Ⅱ/Ⅰ比值减少;Atg5表达也有所减少;而P53蛋白表达明显增加,P均<0.05。相对于常温组,OGD3h,复氧后12 h,亚低温组Rab7,Beclin1,Atg5蛋白表达均高于常温组,P<0.05;而P53蛋白表达低于常温组,P<0.05。Control组、R6h常温组、R6h低温组神经元凋亡率分别为10.2%±3.6%、56.8%±7.6%、20%±6.7%,P<0.05。结论 :原代神经元细胞氧糖剥夺/复氧后,神经元自噬明显减少,凋亡增加;亚低温治疗可促进自噬,减少凋亡。
Objective: To observe the effects of mild hypothermia on apoptosis and autophagy in oxygen-deprived nerve cells. Methods: Cerebral cortical neurons were isolated from newborn SD rats (1 d), and OGD / R model was established. After 3 hours, the cell culture plates were removed from the cell culture box and reoxygenated in 37 ℃ or 34 ℃ incubators for 6 h and 12 h respectively. Western-blot was used to detect the expression of P53 protein, autophagy-related proteins LC3Ⅱ / Ⅰ, Beclin1, Atg5 and Rab7. Tunel method to detect neuronal apoptosis. Results: Neonatal rat primary neurons were isolated, cultured and adhered well 4h after inoculation. At the normal temperature, the primary neurons OGD3h, 6h and 12h after reoxygenation, western-blot Rab7 expression decreased; Beclin1 expression decreased; LC3 Ⅱ / Ⅰ ratio decreased; Atg5 expression also decreased; P53 protein expression increased significantly, P < 0.05. Compared with normal temperature group, OGD3h, and 12 h after reoxygenation, Rab7, Beclin1, Atg5 protein expression in hypothermia group were higher than those in normal temperature group, P <0.05; P53 protein expression was lower than normal temperature group, P <0.05. In Control group, the apoptotic rates of R6h hypothermia group and R6h hypothermia group were 10.2% ± 3.6%, 56.8% ± 7.6%, 20% ± 6.7% respectively, P <0.05. CONCLUSION: After the primary neuronal cells are deprived of oxygen or reoxygenated, autophagy is significantly reduced and apoptosis is increased. Mild hypothermia can promote autophagy and reduce apoptosis.