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ERK是细胞内MAPK信号转导系统的重要蛋白激酶,参与多种刺激的应激反应。本实验建立冷热应激模型,应用免疫组织化学和Western blot方法,研究冷热应激刺激对大鼠脊髓和脊神经节ERK的激活作用。结果显示:(1)正常脊髓非磷酸化ERK1(pan-ERK1)阳性细胞分布于RexedIII-X层,而I-II层没有免疫反应,胞浆着色,没有核膜断裂;磷酸化ERK1/2(p-ERK1/2)阳性细胞见于灰质Rexed各层(I-X),散在分布。免疫产物位于胞浆核周区,少见于细胞核,核膜有断裂。脊神经节p-ERK1/2免疫反应见于大、中、小型神经元,定位于细胞核;没有pan-ERK1免疫反应。(2)20min热应激后,RexedII层p-ERK1/2阳性细胞数明显增多,核周着色区增大,核膜常有断裂,脊神经节阳性细胞数没有明显变化;10min热应激和体温(38℃)应激后,脊髓和脊神经节阳性细胞数和核着色没有显著变化。(3)20min冷应激后,脊髓II层p-ERK1/2阳性细胞数轻度减少,核周着色区变窄,核膜有断裂,脊神经节阳性细胞数没有变化;10min冷应激后阳性细胞数没有明显变化。(4)Western blot显示,热应激和冷应激分别提高和降低了胸段脊髓p-ERK1/2的蛋白表达水平。本研究证实,ERK存在于脊髓灰质各层和脊神经节各型神经元,热应激提高了ERK激活水平而冷应激则相反,提示MAPK信号转导激活具有应激类型和应激强度的依赖性,参与了感觉神经元的功能调节。
ERK is an important protein kinase in intracellular MAPK signal transduction system and is involved in many stimuli of stress response. In this study, we established a model of cold and heat stress, using immunohistochemistry and Western blot method to study the activation of ERK in the spinal cord and spinal ganglion of rats by hot and cold stress. The results showed that: (1) The positive cells of non-phosphorylated ERK1 (pan-ERK1) in normal spinal cord were distributed on RexedIII-X layer, but no immunoreaction, cytoplasm staining and no nuclear membrane rupture in I- p-ERK1 / 2) positive cells were found in all layers (IX) of Rexed gray matter and scattered. Immune products located in the cytoplasm of perinuclear area, rare in the nucleus, nuclear membrane rupture. Spinal ganglia p-ERK1 / 2 immune response seen in large, medium and small neurons, located in the nucleus; no pan-ERK1 immune response. (2) After 20min heat stress, the number of p-ERK1 / 2 positive cells in RexedII layer increased significantly, the perinuclear area increased, the nuclear membrane often ruptured, and the number of positive cells in spinal ganglia had no significant changes. 10min heat stress and body temperature (38 ℃) stress, the number of spinal cord and spinal ganglia positive cells and nuclear staining did not change significantly. (3) After 20min cold stress, the number of p-ERK1 / 2 positive cells in spinal lamina II decreased slightly, the perinuclear pigmented area became narrower, nuclear membrane ruptured and the number of positive cells in spinal ganglia did not change; 10min cold stress was positive The number of cells did not change significantly. (4) Western blot showed that heat stress and cold stress increased and decreased p-ERK1 / 2 protein expression in the thoracic spinal cord respectively. This study confirmed that ERK exists in various layers of spinal cord gray matter and spinal ganglion neurons, heat stress increased activation of ERK and cold stress is the opposite, suggesting that MAPK signal transduction activation has a stress type and stress-dependent Sexual, involved in the functional regulation of sensory neurons.