PKMζ在丰富康复训练改善局灶性脑缺血大鼠认知功能中的作用和机制研究

Cognitive disorder in cerebral ischemic patients will seriously affect their works and lives. Rehabilitation training can improve cognition. Researches showed that rich rehabilitation training can improve neurological function of damage brain, and it is superior to other rehabilitation mode on motor function recovery. So we speculate it is also superior to other rehabilitation mode on cognition recovery. PKMζ is a specific protein kinase which expresses only in brain tissue. This protein kinase played an important role in the process of synaptic morphology change and synaptic signal transmission for its keeps activity. It maintains learning and memory by regulating synaptic plasticity. However, whether it is involved in the process of cognition recovery by rich rehabilitation training is not clear. In this research, recombinant retroviral vectors are constructed to overexpress or suppress PKMζ, and the techniques of molecular biology, laser confocal scanning and patch-clamp are used to explore the effect of rich rehabilitation training on cognition recovery and express of PKMζ at first. Furthermore, we will clarify the mechanism of PKMζ regulating synaptic plasticity and its role in the process of cognition recovery of focal cerebral ischemia which generated by rich rehabilitation training. These findings will provide a experimental evidence support for clinical application of rich rehabilitation training, and give new insight into the treatment of focal cerebral ischemia with cognitive impairment.

脑缺血患者遗留的认知障碍会严重影响工作生活，康复训练可以改善认知功能。目前认为丰富康复训练能改善脑损伤后的神经功能，且对运动功能的恢复要优于其他康复模式，推测其在改善认知功能上也优于单一康复训练。PKMζ是一种特异表达在脑组织的蛋白激酶，因其时刻处于激活状态，在突触形态改变及突触信号传递过程中发挥了重要作用，可以通过调节突触可塑性来影响学习记忆的维持,但其是否参与了丰富康复训练影响的认知功能恢复过程尚不清楚。本项目拟从动物和细胞两个层面，通过构建重组慢病毒干预海马神经元PKMζ基因的表达，采用分子生物学、激光共聚焦、膜片钳等技术，观察丰富康复训练对认知功能及PKMζ表达的影响，探讨PKMζ调节突触可塑性在丰富康复训练改善局灶性脑缺血认知功能中的机制。为临床上丰富康复训练的应用提供理论依据，并为局灶性脑缺血伴认知障碍的治疗提供新的思路。

缺血性卒中引起的认知功能障碍目前仍缺少有效的药物治疗，而近期研究提示丰富康复训练可以改善脑血流量，促进树突生长，改善突触的功能，可能利于空间认知能力的恢复。因此本研究建立了单纯认知障碍的脑缺血大鼠模型，通过丰富康复训练干预，对大鼠进行Morris水迷宫测试，从行为学上评估丰富康复训练对认知功能的影响。PKMζ是一种特异性激活的蛋白激酶，在突触形态改变及突触信号传递过程中发挥了重要作用，本研究采用丰富康复训练的脑缺血大鼠模型，利用Western blot和免疫荧光技术分析突触相关蛋白、PKMζ及相关调节蛋白的蛋白表达变化及分布。膜片钳技术记录海马突触传递过程中的电位变化。研究结果提示：不同康复模式均引起脑缺血大鼠突触相关蛋白、PKMζ及相关调节蛋白随康复时间呈现动态变化，而丰富康复训练可以明显促进脑缺血大鼠海马突触信号的传递，改善脑缺血大鼠的空间学习记忆能力，并且，这种突触传递增强能被PKMζ的抑制剂完全抑制。此外，本研究组已利用大鼠海马元代神经元建立了体外氧糖剥夺模型，这对今后进一步利用体外实验研究PKMζ对突触后膜AMPA 受体的钙通透性影响及分子机制提供了理论有利条件。