Chen J, Graham S H, Nakayama M, Zhu R L, Jin K, Stetler R A, Simon R P
Department of Neurology, School of Medicine, University of Pittsburgh, PA 15213, USA.
J Cereb Blood Flow Metab. 1997 Jan;17(1):2-10. doi: 10.1097/00004647-199701000-00002.
The proto-oncogenes bcl-2 and bcl-x-long have been shown to suppress apoptotic cell death in a variety of in vitro systems and cell lines, including neurons. An alternatively spliced from of bcl-x, bcl-x-short, is a promoter of apoptotic death. Whether these genes are induced after ischemia or play any role in determining the fate of ischemic neurons is unknown. To begin to address this issue, we studied the expression of bcl-2, and bcl-x mRNA and protein after global ischemia in the rat. Ischemia was induced in isoflurane-anesthetized rats by the four-vessel occlusion method. mRNA expression was studied by Northern blot analysis at 24 h after ischemia and by in situ hybridization at 2, 4, 8, 24, and 72 h after 15 min of global ischemia. Protein expression was studied using both immunocytochemistry at 4, 8, 16, 24, and 72 h after ischemia and Western blot analysis from tissue harvested at 16, 24, and 72 h after ischemia. Western blots showed that bcl-x-long is the predominant form of bcl-x protein expressed in both normal and ischemic brain. Both bcl-2 and bcl-x-long mRNA were expressed in CA1, CA3, and the molecular layer of the dentate after ischemia. However, bcl-2 and bcl-x protein were expressed only in CA3 and dentate. Thus, while bcl-2 and bcl-x-long mRNA were expressed in both surviving and dying neurons, their proteins were expressed in neurons destined to survive. These results support potential roles for these two apoptosis suppressor proteins in promoting survival after cerebral ischemia.
原癌基因bcl-2和bcl-x-long已被证明在包括神经元在内的多种体外系统和细胞系中可抑制凋亡性细胞死亡。bcl-x的一种可变剪接形式bcl-x-short是凋亡性死亡的促进因子。这些基因在缺血后是否被诱导，或在决定缺血性神经元的命运中是否发挥任何作用尚不清楚。为了开始解决这个问题，我们研究了大鼠全脑缺血后bcl-2、bcl-x mRNA和蛋白的表达。通过四血管闭塞法在异氟烷麻醉的大鼠中诱导缺血。在缺血后24小时通过Northern印迹分析研究mRNA表达，并在全脑缺血15分钟后的2、4、8、24和72小时通过原位杂交进行研究。在缺血后4、8、16、24和72小时使用免疫细胞化学研究蛋白表达，并在缺血后16、24和72小时从收获的组织进行蛋白质印迹分析。蛋白质印迹显示，bcl-x-long是在正常和缺血脑表达的bcl-x蛋白的主要形式。缺血后，bcl-2和bcl-x-long mRNA在CA1、CA3和齿状回分子层均有表达。然而，bcl-2和bcl-x蛋白仅在CA3和齿状回表达。因此，虽然bcl-2和bcl-x-long mRNA在存活和即将死亡的神经元中均有表达，但其蛋白仅在注定存活的神经元中表达。这些结果支持这两种凋亡抑制蛋白在脑缺血后促进存活中的潜在作用。
