Comella J X, Sanz-Rodriguez C, Aldea M, Esquerda J E
Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, Spain.
J Neurosci. 1994 May;14(5 Pt 1):2674-86. doi: 10.1523/JNEUROSCI.14-05-02674.1994.
The purpose of the experiments reported here is to provide evidence that motoneurons (MTNs) isolated from chick embryo spinal cords go through an active process of cell death when deprived of trophic support in vitro. In order to analyze and characterize this process, MTNs were isolated with a metrizamide gradient technique and cultured in the presence of saturating concentrations of soluble muscle extract. When muscle extract was washed off from the cultures, MTNs entered a process of cell death that could be blocked with inhibitors of mRNA and protein synthesis. Two other additional criteria were used to define this process as an active one. First, ultrastructural analysis of MTNs dying as a consequence of muscle extract deprivation showed that some, but not all, of the MTNs displayed clear signs of apoptotic cell death. Those included cytoplasm condensation, fragmentation of chromatin, and preservation of cytoplasmic organelles. Second, internucleosomal degradation of DNA was detected in MTNs deprived of muscle extract. When DNA was analyzed by Southern hybridization techniques using digoxigenin-labeled genomic probes, a clear ladder pattern could be identified on muscle extract-deprived MTNs. The degradation of DNA upon trophic deprivation could be prevented by cycloheximide (CHX). In an attempt to characterize further the process of active cell death in MTNs, we found a time point of commitment to cell death of approximately 10 hr by using three different approaches: muscle extract deprivation plus readdition of muscle extract, muscle extract deprivation plus addition of CHX, and muscle extract deprivation plus addition of actinomycin D. Moreover, we show that MTNs deprived of trophic support from muscle extract but maintained alive with CHX could not be rescued from cell death by reading muscle extract if CHX was washed off the cultures within the first 15 hr of muscle extract deprivation. However, muscle extract alone was able to rescue MTNs that had been kept alive with CHX for periods of time longer than 24 hr after muscle extract deprivation. From these results we postulate that the activation of the cell death program after trophic deprivation is transient.
本文所报道实验的目的是提供证据表明，从鸡胚脊髓中分离出的运动神经元（MTN）在体外缺乏营养支持时会经历一个主动的细胞死亡过程。为了分析和表征这个过程，采用甲泛葡胺梯度技术分离MTN，并在饱和浓度的可溶性肌肉提取物存在下进行培养。当从培养物中洗去肌肉提取物时，MTN进入细胞死亡过程，该过程可被mRNA和蛋白质合成抑制剂阻断。另外还使用了两个标准来将这个过程定义为主动过程。首先，对因肌肉提取物剥夺而死亡的MTN进行超微结构分析表明，部分（而非全部）MTN显示出凋亡细胞死亡的明显迹象。这些迹象包括细胞质浓缩、染色质碎片化以及细胞质细胞器的保留。其次，在缺乏肌肉提取物的MTN中检测到DNA的核小体间降解。当使用地高辛标记的基因组探针通过Southern杂交技术分析DNA时，在缺乏肌肉提取物的MTN上可以识别出清晰的梯状模式。营养剥夺时DNA的降解可被环己酰亚胺（CHX）阻止。为了进一步表征MTN中主动细胞死亡的过程，我们通过三种不同方法发现细胞死亡的关键时间点约为10小时：肌肉提取物剥夺后再添加肌肉提取物、肌肉提取物剥夺后添加CHX以及肌肉提取物剥夺后添加放线菌素D。此外，我们表明，缺乏肌肉提取物营养支持但用CHX维持存活的MTN，如果在肌肉提取物剥夺的前15小时内将CHX从培养物中洗去，再添加肌肉提取物也无法使其从细胞死亡中挽救回来。然而，单独的肌肉提取物能够挽救在肌肉提取物剥夺后用CHX维持存活超过24小时的MTN。从这些结果我们推测，营养剥夺后细胞死亡程序的激活是短暂的。
