大鼠成年海马神经干细胞体外氧糖剥夺／复氧模型的建立  

Establishment of rat models of oxygen glucose deprivation/reoxygenation in adult neural stem cells in vitro

作　　者：谭盛[1] 陈健[1] 郭阳[1] 陈瑞清[1] 李粲[1] 陈镇洲[2] 

机构地区：[1]广东省脑功能修复与再生重点实验室,南方医科大学珠江医院神经内科,广州510282 [2]广东省脑功能修复与再生重点实验室,南方医科大学珠江医院神经外科,广州510282

出　　处：《中华神经医学杂志》2011年第12期1238-1242,共5页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（30801184）;广东省科技计划重点专项（2011A030400007）

摘　　要：目的探讨一种简便、稳定、可重复的大鼠成年神经干细胞体外氧糖剥夺／复氧模型的制备方法。方法以来自于成年Fisher344大鼠的海马神经干细胞系为研究对象，以无血清培养基培养并传代．并用nestin和DAPI免疫荧光双染确认其生物学特性。将三气培养箱氧气浓度调至1％以制备缺氧环境，将培养基换为不含葡萄糖的Earle’s平衡盐溶液，分别缺氧缺糖2h、4h、6h、8h、10h后取出细胞，恢复正常条件继续培养24h后倒置显微镜下观察细胞形态学变化，CCK-8比色法检测细胞存活率，流式细胞术检测细胞凋亡率。同时设置常氧常糖的正常对照组。结果与正常对照组相比．缺氧缺糖2h组细胞吸光度值明显升高，细胞存活率有一定的增长，但差异无统计学意义（P〉0．05）。随着缺氧缺糖时间延长，神经干细胞形态学损伤逐渐加重，细胞吸光度值逐渐下降．缺氧缺糖6h后与正常对照组比较差异有统计学意义（P〈0．05）；缺氧缺糖6h起细胞存活率均较正常对照组明显下降，比较差异有统计学意义伊〈0．05）；神经干细胞凋亡率逐渐增高，且均明显高于正常对照组，差异有统计学意义（P〈0．05），其中缺氧缺糖6h时细胞的凋亡率已超过50％。结论利用三气培养箱物理缺氧方法可成功建立一种简便、有效的神经干细胞体外氧糖剥夺／复氧模型。Objective To establish simple, stable and reliable rat models of oxygen glucose deprivation/reoxgenation（OGD/R） in adult neural stem cells （NSCs） in vitro. Methods The NSCs from adult Fisher344 rats were cultured in serum-flee medium and identified using nestin and DAN immunofluorescent double staining. These cells were washed with a Earle＇s balanced salt solution without glucose for 2 times, then, incubated for different periods （2, 4, 6, 8 and 10 h） in a trigas incubator with an atmosphere of 1% 02, 5%CO2 and 94% N2, 98% humidity at 37℃. And then, these cells were removed from the anaerobic incubator, washed, and added DEME/F12 containing bFGF supplement. A normoxic-normoglycemic control group was employed. Morphological assessment of NSCs was performed by light microscopy after re-oxgenation for 24 h; CCK-8 colorimetric method was used to determine the survival and proliferation of NSCs, and flow cytometry was employed to detect the apoptosis of NSCs. Results After the setting of oxygen glucose deprivation for 2 h, the OD value and the survival rate in the OGD cells were increased as compared with those in control group without significant difference （P〉0.05）. While the morphological damage of NSCs aggravated gradually and the OD value decreased in OGD cells following the prolongation of times; under the setting of oxygen glucose deprivation for 6 h, the OD value in OGD cells obviously decreased as compared with that in the control group （P〈0.05）; under the setting of oxygen glucose deprivation for 6 h, the survival rate obviously decreased and the apoptosis rate significantly increased in OGD cells as compared with that in the control group （P〈0.05）; under the setting of oxygen glucose deprivation for 6 h, the apoptosis rate of NSCs excessed to 50%. Conclusion By means oftrigas incubator, simple, stable and reliable models of OGD/R in NSCs in vitro can be successfully established.

关 键 词：氧糖剥夺/复氧 神经干细胞 细胞凋亡 实验模型 

分 类 号：R285.5[医药卫生—中药学]