Study on Intracellular Mechanism in Neuronal Apoptosis

胎鼠海马神经元在50%氧气气氛中培养，培养20小时后神经元逐渐死亡。这种死亡模式被发现是由细胞内的主动死亡程序介导的，即所谓的凋亡，如下：1)蛋白质和RNA合成抑制剂放线菌酮和放线菌素-D分别阻止细胞死亡，表明细胞死亡需要新的蛋白质生物合成。2)在细胞死亡过程中检测到DNA片段化(称为DNA梯状条带)，这是一种特异性的细胞凋亡生化标志。3)高K+(26-50 mM)去极化可防止细胞死亡。这种作用可被二氢吡啶类化合物、L类钙通道阻滞剂硝苯地平和尼卡地平所抑制。这些结果表明，细胞内氧代谢的破坏激活了培养的海马神经元的细胞自杀程序，神经元的活动在保护神经元免受TH-…期间的氧化损伤中发挥了重要作用。7-9日龄大鼠小脑颗粒神经元在高K+(26 MM)培养液中体外培养4-5天。然后用低K+(5 MM)的培养液培养，导致大量颗粒神经元死亡。颗粒神经元的死亡以细胞凋亡为特征。在这项研究中，我们利用上述系统研究了各种神经营养因子对神经元存活的影响。我们发现脑源性神经营养因子(BDNF)和神经营养因子-4/5(NT-4/5)对低K+诱导的神经元有保护作用，而神经生长因子(NGF)无此作用。据报道，神经营养素-3(NT-3)对神经元存活的影响很小。为了确定颗粒神经元是否对BDNF有直接反应，我们分析了这些神经元中Fos蛋白的诱导。在暴露于神经色氨酸后合成Fos蛋白的单个细胞可以使用Fos抗体来识别。免疫细胞化学染色显示，较多的小脑颗粒神经元对BDNF呈免疫反应，但在没有BDNF或有NGF存在的情况下，很少有免疫反应。我们的结果表明，BDNF对成熟的小脑颗粒神经元有直接作用，并能保护这些神经元在低K+条件下免于凋亡。较少

When embryonic rat hippocampal neurons were cultured in a 50% oxygen atmosphere, neurons gradually died after 20 hr in culture. This death pattern was found to be mediated by an intracellular active death program, so called apoptosis, as follows : 1) Cycloheximide and actinomycin-D,protein and RNA synthesis inhibitors, respectively, prevented cell death, indicating that cell death requied new protein biosynthesis. 2) DNA fragmentation (called a "DNA ladder" ), a specific biochemical marker of apoptosis, was detected during the course of cell death. 3) Depolarization with high K+ medium (26-50mM) prevented cell death. This effect was suppressed by some dihydropyridine derivatives, L-type Ca channel blockers, such as nifedipine and nicardipine. These results suggested that disruption of intracellular oxygen metabolism activated the cell suicide program in the cultured hippocampal neurons, and that neuronal activity playd an important role in saving neurons from oxidative damage during th … More eir long life without division.Cerebellar granule neurons obtained from 7-9 day-old rats were grown in vitro for 4-5 days in high K+ (26 mM) medium. The culture medium was then with that containing low K+ (5 mM) which caused a large number of granule neurons to die. The death of granule neurons has been characterized as apoptosis. In this study, we investigated the effects of various neurotrophins on neuronal survival using the above system. We found that brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) but not nerve growth factor (NGF) can protect these neurons from apoptosis in low K+. Neurotrophin-3 (NT-3) had a small effect on neuronal survival as reported. To determinewhether the granule neurons respond directly to BDNF,we analyzed the induction of the Fos protein in these neurons. Individual cells that synhtesize Fos protein after exposure to neurotrpohin can be recognized using antibodies to Fos. Immunocytochemical staining of the cultures demonstrated that a relatively large number of cerebellar granule neurons showed immunoreactivity in response to BDNF,but few of them were immunoreactive in the absence of BDNF or in the presence of NGF.Our results suggested that BDNF has a direct effect on mature cerebellar granule neurons and can protect these neurons from apoptosis in low K+. Less