RKIP在工频磁场暴露对阿尔茨海默症发生影响中的作用及机制研究

It has been reported that power frequency magnetic field (MF) exposure may promote the genesis of Alzeimer's disease (AD) in epidemiology investigation.However,the experimental report about the relationship between MF and AD is still unknown. RKIP has been indicated to be participated in the pathological process of AD. Our study found that RKIP was well related with the influence of MF on AD genesis. Based on the importance of the regulation of Ach and apoptosis by RKIP, we postulate that RKIP is critical for the influence of MF on AD. Taking RKIP as the starting point, this study will deeply investigated the influence of MF on AD genesis and its mechanisms. Firstly, the animal model of MF combined with AD will be established, then the influences of MF on cognitive function, hippocampus stucture and bio-markers of AD in AD rats will be explored. Moreover, the changes of Ach synthesis and metabolism,the phosphorylation of RKIP and its apoptotic-related signalling pathways, and protein interaction will be detected. Secondly, the apoptotic cell models of primary hippocampus neurons and PC12 neural cells induced by MF combined with Aβ will be used. HCNP, inhibitor, RKIP overexpression and RNA interference et al will be applied to investigate the role of regulation of Ach and apoptosis mediated by RKIP in the influece of MF on AD. This study will further illuminate the relationship between MF and AD and the molecular mechanisms related to RKIP, and provide a new pathway for prevention and cure.

流行病学调查显示，工频磁场（MF）可能增加阿尔茨海默症（AD）的发病风险，然MF与AD关系的实验研究尚未见报道。研究表明，RKIP参与AD的病理过程。我们研究发现，RKIP与MF对AD发生的影响密切相关。基于RKIP对海马Ach及凋亡调控的重要意义，我们推测RKIP在MF对AD的影响中可能发挥重要作用。本项目拟以RKIP为切入点，深入探讨MF对AD发生的影响及机制。首先，建立MF联合AD动物模型，研究MF对AD大鼠认知功能、海马结构及AD标志物的影响，并检测海马Ach合成与代谢、RKIP磷酸化及其凋亡信号通路以及蛋白相互作用改变；然后,通过MF联合Aβ诱导的原代海马神经元和PC12神经细胞凋亡模型，分别应用HCNP、抑制剂、RKIP过表达、RNA干涉等，研究RKIP介导的Ach及凋亡调控在MF对AD影响中的作用。这将进一步揭示MF与AD的关系，阐明RKIP相关分子机制，并为防治提供新思路。

本项目拟通过工频磁场（Power frequency electromagnetic field， PF-MF）暴露联合阿尔茨海默症（Alzheimer’s diease，AD）大鼠及神经细胞模型，采用行为学、形态学、分子生物学方法，研究PF-MF对AD发生的影响，并进一步探讨Raf激酶抑制蛋白（Raf kinase inhibitor protein，RKIP）及其调控的信号通路在其中的作用。随后采用基因转染方法建立RKIP过表达及低表达的稳转PC12神经细胞模型，明确RKIP在PF-MF暴露对AD发生发展影响中的作用。.研究发现1、一定条件的PF-MF暴露可导致正常动物学习记忆功能和海马形态结构发生可恢复性损伤（50Hz-400uT 60d），并可促进正常神经细胞增殖同时增加其凋亡（50Hz-100uT 24h）。2、一定条件的PF-MF暴露可改善AD动物学习记忆功能障碍，减轻海马形态结构损伤（50Hz-400uT 60d）；促进AD细胞增殖，并抑制其凋亡（50Hz-100uT 24h）。3、PF-MF改善AD病理生理损伤是多因素多途径综合作用的结果，其主要分子机制为：（1）上调SNAP25、UCHL1、EFHD2、MBP表达，促进突触生长并改善神经元损伤；（2）上调RKIP及TAK1表达同时增加二者相互作用，抑制IKK磷酸化，进而抑制由NF-κB通路过度活化所致炎性损伤；（3）下调Caspase3、CytC、Bax表达，并上调Bcl-2表达，抑制细胞凋亡。4、在体外条件下，增加神经细胞RKIP表达，可使其对PF-MF暴露的促增殖作用更为敏感，且能更有效地抵抗Aβ所致增殖活力下降及凋亡增加。.本项目首次以RKIP作为切入点，通过整体动物实验及离体细胞实验，深入了解了PF-MF对AD发生及发展的影响，并探讨了其中的分子机制，具有明显的创新性。本研究结果为进一步明确PF-MF与AD间的关联、阐明AD发生机制提供了实验依据，并为AD防治提供了新思路，具一定理论意义和潜在应用价值。