l-Deprenyl, blocking apoptosis and regulating gene expression in cultured retinal neurons.
l-Deprenyl, blocking apoptosis and regulating gene expression in cultured retinal neurons.
复制标题
L-Deprenyl，阻断培养的视网膜神经元细胞凋亡并调节基因表达。
DOI：
10.1016/s0006-2952(99)00208-7
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发表时间：
1999
影响因子：
5.8
通讯作者：
Ma,JX
中科院分区：
文献类型：
--
作者：
Xu,L;Ma,J;Seigel,GM;Ma,JX
细胞凋亡是许多形式的视网膜变性的最终途径。E1 A-NR 3是一种永生化的视网膜细胞系，表现出视网膜神经元的特定表型。本研究通过血清剥夺和缺氧两种缺血性攻击诱导这些细胞凋亡。结果表明，这两种攻击通过诱导凋亡显著降低活细胞数，如通过活细胞计数、DNA片段化分析和通过TdT介导的dUTP-生物素缺口末端标记（TUNEL）方法原位标记凋亡细胞所揭示的。已知1-丙炔苯丙胺是单胺氧化酶抑制剂，最近发现其具有神经营养活性。我们着手确定l-deprenyl对视网膜细胞的保护作用，并描述其独立于单胺氧化酶抑制的机制。在低至0.0001和0.001 μM的浓度下，l-丙炔苯丙胺分别在无血清和缺氧条件下显著增加存活细胞的数量。在0.001至10 μM范围内，该效应似乎取决于l-丙炔苯丙胺浓度。神经营养活性是通过阻断凋亡，因为l-丙炔苯丙胺在无血清或缺氧条件下减少DNA片段和阳性染色的凋亡细胞的数量。使用mRNA差异显示，9种mRNA被鉴定并通过北方印迹分析证实在暴露于缺氧8小时时具有改变的表达水平。其中5个与GenBank中的任何现有序列都不匹配，而其他4个代表已知基因，包括c-jun，热休克蛋白hsp 70，磷酸甘油酸激酶（PGK）和calpactin I重链。缺氧后4种mRNA表达均显著增加。c-jun，PGK和calpactin mRNA，但不是热休克蛋白70，也增加了血清撤回。l-丙炔苯丙胺部分逆转c-jun和hsp 70 mRNA水平的增加，但不逆转PGK和calpactin的增加。这些结果表明，l-丙炔苯丙胺阻断缺氧诱导的细胞凋亡，以及生长因子撤出和调节凋亡相关基因的表达。
Apoptosis is the final pathway of many forms of retinal degeneration. E1A-NR3 is an immortalized retinal cell line that manifests specific phenotypes of retinal neurons. The present study induced apoptosis in these cells by two ischemic assaults, serum deprivation and hypoxia. The results demonstrated that both the assaults decreased viable cell numbers significantly by inducing apoptosis, as revealed by viable cell count, DNA fragmentation analysis, and in situ labeling of apoptotic cells by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method. l-Deprenyl is known to be a monoamine oxidase inhibitor, and it was found recently to have neurotrophic activities. We set out to determine the protective effect of l-deprenyl on retinal cells and delineate its mechanism independent of monoamine oxidase inhibition. At concentrations as low as 0.0001 and 0.001 μM, l-deprenyl significantly increased the numbers of surviving cells under serum-free and hypoxic conditions, respectively. This effect appeared to be dependent upon the l-deprenyl concentration within the range of 0.001 to 10 μM. The neurotrophic activity was via blocking apoptosis, as l-deprenyl decreased the fragmented DNA and the numbers of positively stained apoptotic cells under serum-free or hypoxic conditions. Using mRNA differential display, nine mRNAs were identified and confirmed by northern blot analysis to have altered expression levels at 8 hr of exposure to hypoxia. Five of them do not match any existing sequences in GenBank, whereas the other four represent known genes including c-jun, heat-shock protein hsp70, phosphoglycerate kinase (PGK), and calpactin I heavy chain. All of the four mRNAs were increased significantly by hypoxia. The c-jun, PGK, and calpactin mRNAs, but not hsp70, also were increased by serum withdrawal. l-Deprenyl partially reversed the increase in c-jun and hsp70 mRNA levels, but not in PGK and calpactin. These results suggest that l-deprenyl blocks apoptosis induced by hypoxia as well as by growth factor withdrawal and regulates the expression of apoptosis-related genes.