基于miRNA时空特异性调控Wnt通路的益气活血通络法影响神经再生机制研究

The expression of miRNA in brain tissue with time-space specificity in regulating Wnt singal paths.So,it influences the mechanism of nerve regeneration,which is acknowledged as one of the mechanisms of nerve regeneration related to gene regulation network.Naoluoxintong,the representative prescription of the method of tonifying Qi, promoting blood flow and dredging collatera from Wang medicine of Xinan medical has an exact curative effect.The study takes the effect of the expression of miRNA on relevant signal molecule as the starting point to put forward the scientific hypothesis that the expression of the time-space specificity of miRNA, participates in the regulatory mechanism of the nerve regeneration because of Qi-deficiency and blood stasis in cerebral and explore the intervention effect of the method of tonifying Qi, promoting blood flow and dredging collatera.The multi-factor composite simulation model of Qi-deficiency and blood stasis of MCAO/R is made. Through the dynamic comparision,studies both in vivo and in vitro,by the means of laser scanning confocal microscopes,Laser Doppler Flowmeitry,Morris Water Maze,LNA-ISH,Western-blot,real-time PCR and immunofluorescence,the researchers observe the distinctive expression and changes of miRNA in different cerebral region, Wnt singal paths,the proliferation and differentiation of NSC,rCBF and the spatial cognitive function,and explore the regulating mechanism of miRNA to elementarily clarify pathologic nature of the model and therapeutic mechanism of the therapy，and provide experimental evidences for the development and clinical application of the method of tonifying Qi, promoting blood flow and its representative prescription.

脑组织miRNA时空特异性表达调控Wnt信号通路而影响神经再生的机制，已被认为是神经再生过程基因调控网络的重要机制之一；“脑络欣通”作为新安王氏内科益气活血通络法代表方对缺血性中风有确切疗效。本研究以miRNA对相关信号分子的影响为切入点，提出“不同脑区miRNA时空特异表达参与气虚血瘀脑缺血神经再生的调控机制”假说，观察益气活血通络法干预作用。多因素复合模拟制作气虚血瘀MCAO/R模型，动态比较、在体和离体结合研究方式，借助激光共聚焦、激光多普勒仪、MWM、LNA-ISH、Western blot、real-time PCR及免疫荧光化学等技术方法，观测不同脑区miRNA、Wnt通路关键分子、NSC增殖分化、rCBF及空间认知功能的特征性表达和变化，探索miRNA调控机制，初步阐明该模型病理本质及该治法的干预机制，为益气活血通络法及其代表方的开发和临床应用提供实验依据。

脑组织miRNA特异性表达调控Wnt通路而影响神经再生的机制是神经再生过程基因调控网络的重要机制之一；“脑络欣通”作为新安医学益气活血通络法代表方对缺血性中风有确切疗效，有确切的中医理论指导基础。本研究以miRNA对相关信号分子的影响为切入点，提出“不同脑区miRNA时空特异表达参与气虚血瘀脑缺血神经再生的调控机制”假说，观察益气活血通络法干预作用。多因素复合模拟制作气虚血瘀MCAO/R模型，动态比较、在体和离体结合研究方式，借助激光多普勒仪、MWM、LNA-ISH、Western blot、real-time PCR及免疫荧光化学等技术方法，观测不同脑区miRNA、Wnt通路关键分子、NSC增殖分化、rCBF及空间认知功能的特征性表达和变化，探索miRNA调控机制，同时在相关新安医学特色中医理论研究的指导下，初步阐明该模型病理本质及该治法的干预机制，为益气活血通络法及其代表方的开发和临床应用提供实验依据。. 实验结果发现，本项目研制的MCAO/R气虚血瘀证大鼠模型是在符合脑缺血现代医学客观评价标准基础上，同时采用饥饿、疲劳、低氧、高脂饮食、高龄等多因素复合制作出的同时具备气虚血瘀证候特点的病证结合实验模型。结合项目固本培元对益气活血理论指导的研究，益气活血通络法可通过调节特定miRNA干预Wnt通路继而影响脑缺血后的神经再生过程。为miRNA“时空特异性”表达参与的复杂信号通路分子生物学机制的阐明和“病证结合模型”分子生物学水平标准化的研究提供基础和启发，为今后深入系统研究基于miRNA参与调控的益气活血通络法治疗脑缺血作用的现代科学内涵打下基础。