缺氧条件下葡萄糖对成年神经干细胞体外增殖影响的研究

Effects of glucose concentration on proliferation of hypoxic adult neural stem cells(ANSCs) in vitro

目的探讨不同浓度的葡萄糖对氧/糖剥夺条件下体外成年大鼠神经干细胞活力和增殖的影响。方法无血清培养成年Fisher 344大鼠海马神经干细胞系,用Nestin和DAPI免疫荧光双染以确认其生物学特性。将三气培养箱氧气浓度调至1%以制备缺氧环境,将培养基换为无糖Earle’s平衡盐溶液缺氧培养6 h后更换为含有不同浓度葡萄糖(7.5、17.5、27.75、41.75、83.75 mmol/L)的培养基恢复正常条件继续培养后,检测神经干细胞的活力和形态学改变。同时设置常氧常糖为正常对照组和甘露醇为渗透压对照组。结果 CCK-8检测显示,常糖常氧正常对照组细胞吸光度值较缺氧模型组高,7.5 mmol/l糖培养组以及41.75 mmol/L和83.75 mmol/L糖培养组吸光度值均较正常糖缺氧组(17.5 mmol/L)低,差异有统计学意义(P<0.05);而27.75 mmol/L糖培养组吸光度值较正常糖缺氧组低,差异无统计学意义,且排除了高糖培养基引起的渗透压力的影响。各组细胞形态学改变与CCK-8检测结果一致。结论缺氧后轻度升高的糖对体外缺氧成年神经干细胞的损伤可能有保护作用,而较低浓度和更高浓度的葡萄糖则加重其损伤。

Objective To explore the effects of glucose concentration on the viability and proliferation of hypoxic adult neural stem cells（ANSCs） after OGD.Methods The ANSCs cell line from adult Fisher 344 rats were cultured in serum-free medium and identified using Nestin staining.Anoxic cultured ANSCs（1% O2,94%N2,and 5%CO2 for 6 h） were treated with different concentrations of D-glucose（7.5,17.5,27.75,41.75,83.75 mmol/L）.A normoxic-normoglycemic control group was also employed.CCK-8 colorimetric method was used to determine the survival and proliferation of ANSCs,as well Hoechst 33342 immunofluorescent staining was used to detect the apoptosis of ANSCs.Mannitol was used as a control to exclude a possible effect of osmolality on cell viability.Results Compared with those of the normoxic-normoglycemic control group,the O.D.at 450 nm of OGD group was higher significantly.Furthermore,there was a significant cell viability decrease in cultures exposed to 7.5 mmol/L,41.75 mmol/L and 83.75 mmol/L glucose after hypoxia compared to that of control（P0.05）.There was no difference between 27.75 mmol/L and 17.5 mmol/L glucose groups,under the exclusion of the influence of osmolarity on cell viability.The CCK-8 detection results were consistant with that of microscopic observation.Conclusions Mildly elevated glucose concentration after hypoxia may protect NSCs against hypoxia.