神经生长因子(NGF)对H_2O_2诱导胶质瘤A-172细胞凋亡的抑制效应及其机制(英文)

Prevention and mechanism of H_2O_2-induced apoptosis by nerve growth factor in glioma cell line A-172

目的 :研究神经生长因子 (NGF)对过氧化氢 (H2 O2 )诱导神经胶质瘤细胞凋亡的抑制作用及其机制。方法 :抑制细胞增殖的检测采用MTT法 ,吖啶橙 (AO)染色荧光显微观察细胞的形态学变化 ,DNA琼脂糖电泳检测DNA断裂 ,二硫硝基苯甲酸 (DTNB)法检测细胞内还原型谷胱甘肽 (GSH)水平的变化。结果 :10 0～ 80 0μmol/L的H2 O2 明显抑制A 172细胞增殖 ,2 0 0 μmol/LH2 O2 作用 12h后 ,形态学上表现为染色体聚集、核固缩和断裂 ,电泳可见DNA断裂形成的阶梯状条带。经NGF作用 2 4h后可引起A 172细胞内GSH水平上升近 2倍 ,但NGF抑制H2 O2 诱导A 172细胞凋亡的效应与GSH水平无关 ,而且此过程不需新蛋白或RNA的合成。结论 :H2 O2 可有效诱导神经胶质瘤A 172细胞凋亡 ,NGF对此表现显著抑制效应 ,但与细胞内GSH水平变化无关。

Objective:To investigate the protective effects of nerve growth factor (NGF) on hydrogen peroxide (H 2O 2) induced apoptosis of glioma cell line A 172 and its mechanism Methods:Inhibition of proliferation was measured with a colorimetric 3 [4,5 dimethylthiazol 2 yl] 2,5 diphenyltetrazolium bromide (MTT) assay Morphological assessment of apoptosis was performed with acridine orange(AO) stained fluorescence microscope DNA fragmentation was assessed by agarose gel electrophoresis The level of intracellular glutathione (GSH) was measured with a colorimetric 5,5 dithio bis(2 nitrobenzoic acid)(DTNB) assay Results:Exposure of exponentially growing A 172 cells to H 2O 2 100～800 μmol/L for 12 h resulted in growth arrest After treatment of A 172 cells with H 2O 2 200 μmol/L for 12h,marked morphological changes including “apo bodies”,chromatin condensed and fragmentation were observed with AO stained microscope Agarose gel electrophoresis of DNA from cells treated with 200 μmol/L H 2O 2 for 12 h revealed “ladder”pattern Treatment with NGF for 24 h increased the level of cellular antioxidant glutathione (GSH) by ～2 0 fold However,NGF protected cells against H 2O 2 stress even when cellular GSH was depleted by treatment with L buthionine (S,R) sulfoximine (BSO) The GSH independent protection effects of NGF did not require new protein or RNA synthesis Conclusion:H 2O 2 induces apoptosis of A 172 cells,and that NGF prevent apoptosis independently of level of intracellular GSH