髓鞘碱性蛋白神经毒性的机制研究

发布时间：2018-12-14 11:10

【摘要】：背景：中枢神经系统损伤后会导致髓鞘降解,这不仅抑制神经元生长,甚至诱导神经元变性死亡。脱髓鞘后,髓鞘相关蛋白(Nogo, MAG, OMgp和MBP)将暴露于组织外,产生病理影响。目前对Nogo, MAG, OMgp等"myelin inhibitor"通过Nogo或PirB受体抑制神经再生的研究较为深入具体。生理情况下,MBP与质膜内表面紧密结合,维持正常的髓鞘形态和结构。髓鞘解体后,MBP由胞内脂相环境暴露于胞外水相环境,部分MBP从髓鞘解脱以游离形式存在于细胞外基质中而起作用。长期以来,脱髓鞘病变后,MBP对神经元是否有直接作用仍未清楚。目的：我们发现MBP在体外具有选择性神经毒性作用。本课题拟进一步确认MBP的神经毒性作用,筛选鉴定与其相互作用的膜组分并阐明其作用机制。此研究将有助于我们更好的理解脱髓鞘导致神经元损伤的机制,为今后的临床治疗提供有力指导。方法：我们利用DAPI-PI双染鉴定神经元死亡情况；利用免疫细胞化学方法判断MBP的结合方式；利用蛋白免疫印迹,电生理,钙成像,钙黄绿素通透,人工脂质体分析等实验去探索MBP引起神经元死亡的机制。结果：MBP可以特异地结合到体外培养的神经元表面并特异地诱导神经元死亡。MBP结合到神经元表面具有碱性依赖性,因为这种结合可以被酸性磷脂阻断并受到其他碱性蛋白的竞争性阻断。进一步的研究表明,MBP通过去极化神经元膜、增加对离子和大分子的通透、降低神经元膜的流动性来损伤神经元膜的功能并破坏其完整性。最后,人工脂质体实验显示MBP可以结合并损毁含有酸性磷脂的脂质体。结论：我们的研究表明MBP可以特异性地与神经元膜表面某种酸性成分直接结合,这种结合直接损伤了神经元膜的结构和功能的完整性,从而导致神经元死亡。这些结果提示MBP可能参与了脱髓鞘后的病理过程。
[Abstract]:Background: central nervous system injury leads to myelin degradation, which not only inhibits the growth of neurons, but also induces degeneration and death of neurons. After demyelination, myelin associated proteins (Nogo, MAG, OMgp and MBP) are exposed to tissue and have pathological effects. At present, the inhibition of nerve regeneration by Nogo or PirB receptors in myelin inhibitor such as Nogo, MAG, OMgp is more in-depth and specific. Under physiological conditions, MBP is tightly bound to the inner surface of the plasma membrane to maintain normal myelin morphology and structure. After the demyelination of the myelin sheath, MBP was exposed to the extracellular aqueous environment from the intracellular lipid environment, and partial MBP existed in the extracellular matrix in the form of dissociation from the myelin sheath. For a long time, it is not clear whether MBP has direct effect on neurons after demyelinating lesion. Objective: we found that MBP has selective neurotoxicity in vitro. The purpose of this study is to confirm the neurotoxicity of MBP, to identify the membrane components interacting with MBP and to elucidate its mechanism. This study will help us to better understand the mechanism of demyelination resulting in neuronal injury and provide useful guidance for clinical treatment in the future. Methods: DAPI-PI double staining was used to identify the death of neurons, immunocytochemistry was used to determine the binding mode of MBP. The mechanism of neuronal death induced by MBP was explored by Western blot, electrophysiology, calcium imaging, calcium xanthophyllin permeability and artificial liposome analysis. Results: MBP could specifically bind to the surface of cultured neurons and specifically induce neuronal death. The binding of MBP to the surface of neurons was alkaline dependent. Because this binding can be blocked by acid phospholipid and by other basic protein competitive blocking. Further studies have shown that MBP damages the function and integrity of neuronal membranes by depolarizing neuronal membranes, increasing the permeability of ions and macromolecules, and reducing the fluidity of neuronal membranes. Finally, artificial liposome experiments showed that MBP could bind and destroy liposomes containing acidic phospholipids. Conclusion: our results suggest that MBP can specifically bind to some acidic component on the surface of the neuron membrane, which directly impairs the integrity of the structure and function of the neuron membrane and leads to the death of the neuron. These results suggest that MBP may be involved in the pathological process after demyelinating.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R741

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