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大鼠视神经与坐骨神经的实时定量PCR比较研究:神经调节蛋白-1 mRNA水平的测定

A real-time quantitative PCR comparative study between rat optic and sciatic nerves: determination of neuregulin-1 mRNA levels.

作者信息

Martínez J C, Malavé C, Bosch I, Castillo C, Núñez J, Villegas G M, Villegas R

机构信息

Instituto de Estudios Avanzados (IDEA), Apartado 17606, Caracas 1015-A, Miranda 1080, Venezuela.

出版信息

Brain Res Mol Brain Res. 2004 Nov 4;130(1-2):49-60. doi: 10.1016/j.molbrainres.2004.07.007.

Abstract

Injured axons from peripheral nervous system (PNS) possess the ability to regenerate. In contrast, regeneration of injured axons does not occur in the central nervous system (CNS) or occurs to a limited extent. Previous works have shown that rat sciatic nerve conditioned medium (CM) produced PC12 cells neuronal-like differentiation and neurite outgrowth. In the present work, we compared the expression of neuregulin-1s (NRG-1s) from rat sciatic and optic nerves as members of the PNS and CNS, respectively. Sciatic nerve CM showed a higher neurotrophic activity on PC12 cells than rat optic nerve CM. RT-PCR analysis verified the presence of all three types of NRG-1 mRNAs and their receptors in both types of nerves. Real-time quantitative PCR (QPCR) assays showed that the relative expression levels of all three types of NRG-1 mRNAs were higher in optic nerves than in sciatic nerves. Eleven-day cultured optic nerves showed an increased in NDF and SMDF when compared to freshly isolated optic nerves, whereas GGF decreased. However, 11-day-cultured sciatic nerves only showed an increase in SMDF mRNA. Western blots corroborated the differences in NRG-1 expression profile for both types of nerves and their CMs. Incubation of both CMs with the anti-pan-NRG-1 antibody showed that the neurotrophic activity of the optic nerve CM increased, whereas the sciatic nerve CM remained unchanged. These results indicated that different NRG-1 levels are expressed upon nerve degeneration and the balance between those levels and other neurotrophic factors could have an important role on nerve regeneration.

摘要

外周神经系统(PNS)中受损的轴突具有再生能力。相比之下,受损轴突在中枢神经系统(CNS)中不会发生再生,或者再生程度有限。先前的研究表明,大鼠坐骨神经条件培养基(CM)可使PC12细胞发生神经元样分化并促进神经突生长。在本研究中,我们比较了分别作为PNS和CNS成员的大鼠坐骨神经和视神经中神经调节蛋白-1s(NRG-1s)的表达。坐骨神经CM对PC12细胞的神经营养活性高于大鼠视神经CM。RT-PCR分析证实两种神经中均存在所有三种类型的NRG-1 mRNA及其受体。实时定量PCR(QPCR)检测表明,视神经中所有三种类型的NRG-1 mRNA的相对表达水平均高于坐骨神经。与新鲜分离的视神经相比,培养11天的视神经中NDF和SMDF增加,而GGF减少。然而,培养11天的坐骨神经仅显示SMDF mRNA增加。蛋白质免疫印迹法证实了两种神经及其CM在NRG-1表达谱上的差异。两种CM与抗泛NRG-1抗体孵育后,视神经CM的神经营养活性增加,而坐骨神经CM保持不变。这些结果表明,神经退变时会表达不同水平的NRG-1,这些水平与其他神经营养因子之间的平衡可能对神经再生具有重要作用。

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