神经生长因子对大鼠脑皮质神经细胞凋亡的影响

Nerve growth factor prevents apoptosis induced by N methyl D aspartate and hypoxia/hypoglycemia in cultured cerebral cortical neurons

目的研究神经生长因子（ＮＧＦ）对大鼠脑皮质神经细胞凋亡的影响。方法用原代培养的大鼠胎鼠脑皮质细胞，建立了Ｎ－甲基－Ｄ－门冬氨酸（ＮＭＤＡ）、缺氧／缺糖诱导的神经细胞凋亡模型，并观察了ＮＧＦ的保护作用。结果ＮＭ－ＤＡ３００μｍｏｌ·Ｌ－１与细胞作用２０ｍｉｎ及细胞缺氧／缺糖培养１０ｈ可诱导细胞凋亡。提取细胞ＤＮＡ进行琼脂糖凝胶电泳呈现典型的ＤＮＡ梯状条带，应用流式细胞仪检测，在Ｇ１峰前出现一个“Ｇ１亚峰”，凋亡细胞所占比例为５０．２％及４５．７％，同时出现细胞核染色体聚集、断裂及细胞空泡样变等形态变化。ＮＧＦ（５０，１００μｇ·Ｌ－１）能使ＤＮＡ梯状电泳条带不明显或消失，降低凋亡细胞数，并使细胞超微结构损伤减轻或基本恢复正常。结论ＮＧＦ能拮抗ＮＭＤＡ及缺氧／缺糖诱导的神经细胞凋亡。

AIM To study protective effect of nerve growth factor(NGF) on apoptosis induced by N methyl D aspartate (NMDA) and oxygen/glucose deprivation. METHODS Primary cerebral cortical cultures from rat fetus were used to observe effects of NGF on the inhibition of apoptosis. RESULTS NMDA 30 μmol·L -1 exposured to cortical cell cultures for 20 min or oxygen/glucose deprived cultures for 10 h elicited typical apoptosis biochemical and morphological changes including condensed chromation, nuclear fragmentation, and reduction in volume and DNA degradation by endonucleases into fragments in oligonucleosomes of 180￣200 bases pairs. In addition, the characteristic apoptotic DNA peak fo the cells (i.e. sub G 1 peak) was revealed by flow cytometry. Apoptotic cell ratio was 50 2% and 45 7%, respectivelly. In agarose gel electrophoresis, the DNA extracted from cultured cerebral cells treated with NMDA (300 μmol·L -1 ) or hypoxia/hypoglycemia showed a typical internuclesosmal DNA degradation, i.e. DNA ladder. Previously incubating with NGF (50,100 μg·L -1 ) inhibited NMDA or hypoxia/hypoglycemia induces apoptosis of cultures rat cerebral cells. CONCLUSIONS NMDA(300 μmol·L -1 ) or hypoxia/hypoglycemia induced apoptosis in cerebral cortical cultures and NGF reduced apoptosis.