糖氧剥夺/再灌注促进L2.3神经干细胞内源性Huwe1蛋白表达

Oxygenand Glucose Deprivation/Reperfusion Can Induce Expression of Huwe1 ProteinIn Neural Stem L2.3 CellAnd Activate Endogenous Huwe1 Signaling Pathway

目的建立L2.3神经干细胞糖氧剥夺/再灌注损伤模型,探讨糖氧剥夺/再灌注是否可以促进L2.3神经干细胞内源性Huwe1蛋白表达。方法将L2.3神经干细胞糖氧剥夺6h,根据不同组别再灌注培养8h、16、24h,建立L2.3神经干细胞/再灌注损伤模型。将体外培养的L2.3神经干细胞分成5组:正常对照组(control组)、糖氧剥夺组(OGD)、糖氧剥夺/再灌注8h组(OGD6h+R8h组)、糖氧剥夺/再灌注16h组(OGD6h+R16h组)、糖氧剥夺/再灌注24h组(OGD6h+R24h组)。Control组:该组L2.3神经干细胞不进行糖氧剥夺处理。OGD组:该组L2.3神经干细胞仅仅进行糖氧剥夺6h。OGD6h+R8h组:该组L2.3神经干细胞经历OGD6h后再灌注培养8h。OGD6h+R16h组:该组L2.3神经干细胞经历OGD6h后再灌注培养16h。OGD6h+R24h组:该组L2.3神经干细胞经历OGD6h后再灌注培养24h。结果与Control组比,OGD组L2.3神经干细胞糖氧剥夺6h后,活细胞比例明显减少[(94.15±3.59)vs(56.05±5.01),P<0.01];与OGD前比,OGD6再灌注8h Huwe1蛋白表达开始增加,OGD6h再灌注16h和OGD6h再灌注24h Huwe1蛋白表达量显著增加,Huwe1蛋白表达的条带明显变粗变浓,OGD6h再灌注16h最明显。结论糖氧剥夺可引起L2.3神经干细胞损伤;糖氧剥夺/再灌注可以促进L2.3神经干细胞内源性Huwe1蛋白表达。

Objective To establish oxygen and glucose deprivation and reperfusion（ OGD/R） model of L2.3 neural stem cells in vitro,and teste huwe1 expression in L2. 3 neural stem cells with OGD exposure for 6 hr,followed by reperfusion ceded at the indicated time points,and investigate whether Huwe1 is involved in OGD exposure. Methods The OGD exposure was established by using suger-free culture medium and the three gas incubator filled with 93%N2,5%CO2and 2%O2. After OGD expose for6 hr,the L2. 3 neural stem cells were then cutured in serum-free Medium and reoxygenation for different time（ 8hr、16hr、24hr） respectively. At different time（ before OGD、OGD6h、OGD6h followed by reperfusion 8h、OGD6h followed by reperfusion 16h、OGD6h followed by reperfusion 24h）,the expression of Huwe1 protein in L2. 3 neural stem cells were detected by western blot technology.The L2. 3 neural stem cell were exposed OGD for 6 hour and then the neuronal injury was measured by the viability of the L2. 3neural stem cells.Results Oxygen and glucose deprivation for 6h can decreased the neuronal survival rate to（ 56. 05±5. 01） % of that before OGD. Compared with the controls,the OGD / reperfusion-treated L2. 3 neural stem cells exhibited an increase in Huwe1 protein expression and increase peak of the expression of huwe1 protein appeared 16 h after reperfusion.Conclusions Oxygen and glucose deprivation / reperfusion can induce expression of huwe1 protein and activate endogenous huwe1 signaling pathway.