论文部分内容阅读
目的Munc18被普遍认为存在于神经元细胞内,参与神经递质的释放。虽然已有研究表明在Rasmussen’s脑炎病人中有Munc18自身抗体的产生,但对于Munc18的自身免疫机制目前还不清楚。本研究旨在观察Munc18是否可由神经元释放及是否部分分布于神经元细胞表面,并对Munc18抗体是否能诱导神经元损伤进行研究。方法分离胎龄17天Sprague-Dawley胎鼠大脑皮层神经元,用Neurobasal培养基培养。培养液中的蛋白经三氯乙酸沉淀后进行免疫印迹检测,细胞表面蛋白则用EZ-Link-sulfo-NHS-LC-Biotin进行生物素标记,细胞裂解后用结合有亲和素的琼脂糖珠获取生物素标记的蛋白,随后进行免疫印迹检测。在4°C进行活细胞免疫荧光染色,观察神经元细胞表面Munc18的分布。用乳酸脱氢酶(lactate dehydrogenase,LDH)释放指标检测神经元损伤。结果用免疫印迹法可在神经元培养的条件培养液中检测到Munc18蛋白。用谷氨酸(50μmol/L)处理神经元1 h后,培养液中Munc18含量显著增加,同时培养液中未检测到β-actin和syntaxin1,LDH释放也未明显增加。此外,谷氨酸处理能增强神经元表面的Munc18分布。活细胞免疫荧光染色检测到在谷氨酸处理后,神经元表面分布有Munc18,主要位于神经突起与另一神经元接触的部位以及不同神经元的神经末梢交汇部位。谷氨酸诱导的神经元表面Munc18分布增加可被NMDA受体拮抗剂MK801抑制,而AMPA受体拮抗剂未有明显的抑制作用。LDH检测显示,在培养液含有血清成份的情况下,与对照的c-Fos抗体相比,Munc18抗体可明显诱导神经元损伤。结论一部分Munc18可由神经元释出或在神经细胞表面分布。谷氨酸可通过兴奋NMDA受体促进Munc18的释放和在神经细胞表面的分布。并且,Munc18抗体诱导的神经元损伤依赖于血清中的因子。
Objective Munc18 is generally believed to exist in neurons and participate in the release of neurotransmitters. Although studies have shown that Munc18 autoantibodies occur in Rasmussen’s encephalitis patients, the mechanism of autoimmunity of Munc18 is unclear. The purpose of this study was to investigate whether Munc18 can be released from neurons and partially distributed on the surface of neurons, and to investigate whether Munc18 can induce neuronal damage. Methods Sprague-Dawley fetal rat cerebral cortical neurons isolated from gestational age of 17 days were cultured in Neurobasal medium. The protein in the culture medium was precipitated with trichloroacetic acid and subjected to Western blotting. Cell surface proteins were biotinylated with EZ-Link-sulfo-NHS-LC-Biotin and lysed with avidin-agarose beads Biotinylated protein was obtained, followed by Western blotting. Living cells were immunofluorescently stained at 4 ° C to observe the distribution of Munc18 on the surface of neurons. Neuronal injury was detected using lactate dehydrogenase (LDH) release index. Results Munc18 protein was detected by Western blotting in conditioned medium of neuron culture. After treated with glutamic acid (50μmol / L) for 1 h, the content of Munc18 in the culture medium increased significantly, while β-actin and syntaxin1 were not detected in the culture medium, and the release of LDH did not increase significantly. In addition, glutamate treatment increased Munc18 distribution on neuronal surfaces. Immunofluorescent staining of living cells detected that Munc18 was distributed on the surface of neurons after glutamate treatment, mainly located in the contact area between neurites and another neuron and the junction of nerve endings of different neurons. Increased Munc18 distribution on glutamate-induced neuronal surface was inhibited by NMDA receptor antagonist MK801, while AMPA receptor antagonist had no obvious inhibitory effect. LDH assay showed that Munc18 antibody significantly induced neuronal damage compared to control c-Fos antibody in the presence of serum in culture broth. Conclusion A portion of Munc18 can be released by neurons or distributed on the surface of nerve cells. Glutamate promotes the release of Munc18 and the distribution on the surface of nerve cells by excitatory NMDA receptors. Moreover, Munc18 antibody-induced neuronal damage is dependent on serum factors.