基于Neuroligins-PSD-95信号通路研究电针促进Fmr1基因敲除小鼠海马CA1区突触可塑性改善的机制

By the end of 2012, Nature magazine have deemed confirmed Neuroligins neurons protein can lead to abnormal expression autism symptoms, and fragile X syndrome (FXS) and is closely related to autism gene. Previous research shows that Neuroligins for synaptic function has the regulation effects.Our research shows that: acupuncture treatment is in effect on mental retardation, and electroacupuncture can promote Fmr1 knockout mice hippocampus CA1 area synaptic plasticity change. We using immunohistochemistry 、Western Blot and RT- PCR technology to test protein and gene expression on Fmr1 knockout mice hippocampal CA1 area NRXN1、NL1、NL2、NL3、PSD-95、SAPAAP、p- PSD-95、CREB、p- CREB, using BDS and MSP technical test NL1 NL2 NL3 and PSD-95 methylation modification situation,then analyse the effect on Neuroligins-PSD-95 signal path by electric acupuncture; On this basis, further analysis electroacupuncture whether through the phosphorylation and methylation way to affect Neuroligins-PSD-95 signal path, and then analyzes the mechanism on electroacupuncture promote Fmr1 knockout mice hippocampal CA1 area synaptic plasticity improvement.

2012年底，Nature杂志相继发文证实对突触功能具有调控作用的Neuroligins神经元蛋白的异常表达可导致孤独样症状及脆性X综合征（FXS）与孤独症基因紧密关联。我们前期研究表明临床针刺治疗精神发育迟滞有效且电针可促进Fmr1基因敲除小鼠（广泛用于FXS的研究）海马CA1区突触可塑性的改善。本课题采用免疫组织化学、Western Blot和RT-PCR技术检测Fmr1基因敲除小鼠海马CA1区NRXN1、NL1、NL2、NL3、PSD-95、SAPAAP、p- PSD-95、CREB、p- CREB蛋白或基因的表达情况，采用BDS 和MSP 技术检测NL1、NL2、NL3和PSD-95启动区的甲基化修饰情况，探讨电针是否通过磷酸化和甲基化的修饰方式对Neuroligins-PSD-95信号通路活性变化产生良性影响从而促进突触可塑性改善。

本项目选用FMR1基因（fragile X mental retardation 1，脆性X智力迟钝蛋白1）敲除小鼠作为研究对象，观察电针“长强”穴对FMR1基因敲除小鼠学习记忆能力及Neuroligins-PSD-95信号通路的影响，并进一步观察其对CREB和p-CREB蛋白的影响，探索电针“长强”穴治疗精神发育迟滞（MR）的机制。研究结果表明：（1）电针“长强”穴可改善FMR1基因敲除小鼠学习记忆能力；（2）电针“长强”穴可上调FMR1基因敲除小鼠海马CA1区NLGN-3和PSD-95蛋白的表达；（3）初步研究结果表明，电针“长强”穴可上调FMR1基因敲除小鼠海马CA1区CREB和p-CREB表达，提示针刺长强穴可能是通过磷酸化的修饰方式调控Neuroligin-PSD-95通路，从而对FMR1基因敲除小鼠海马CA1区域突触可塑性的产生影响，进而改善FMR1基因敲除小鼠学习记忆能力，其机制有待进一步研究；（4）电针“长强”穴可上调FMR1基因敲除小鼠海马CA1区域γ-氨基丁酸A型受体α1亚基表达。以上结果为探讨针刺“长强”穴治疗精神发育迟滞的作用机制提供了一定的理论基础。