针刺调节小胶质细胞TLR4通路改善脑缺血损伤机制的分子影像学研究

TRL4 in Tolls receptors are the key switches of microglias activation in the cerebral ischemic situation, and it is the symbol of the inflammatory response after cerebral infarction. Acupuncture therapy can alleviate the inflammatory response after cerebral infarction. However, it is still unclear whether the effect is taken by the activation of microglias and regulation of TLR4 pathway.The applicant and his colleages have already established the right middle cerebral artery occlusion (MCAO) models of rats and carried out the pre-experimental MRI molecular study on ultrasmall superparamagnetic particles of iron oxide (USPIO)with fluorescence contrast materials labling the microglias in vivo. In this research, TLR4 KO MCAO mices are established, MR molecular imaging scans of USPIO labling the microglias are undergone. The number of the infiltrating microglias in the ischemic areas with activative regulation and the spatial and temporal variation characteristics by acupuncturing are systematically studied in 12h, 24h, 48h and 72h time points after acupuncture. The effection of acupuncture regulating the microglia activation in ischmeic cerebral lesions are verified by the number of microglias and their activated state with double staining of microglias and iron particles, and by the analysis on the change of TLR4 pathway from the methods of real time PCR and Wester-blot quantitative studies. The relationship between the changes of TLR4 pathway and the MRI signal of USPIO which labling the mircoglias in the activative situation is analyzed. The mechanism of acupuncture regulating the activation of microglias and the TLR4 pathway of cerebral ischemia is explored and demonstrated. Molecular imaging evidences may be provided and verified that the traditional acupuncture can relieve the inflammatory response after cerebral infarction.

Toll样受体TRL4是脑缺血后小胶质细胞激活重要开关，是脑缺血炎症发生标志。针刺可减轻脑梗死后炎性反应，可能机制是通过调节小胶质细胞活化及对TLR4信号通路的调控来实现。申请人预实验已在鼠中动脉梗塞模型上进行了USPIO标记内源性小胶质细胞的活体MR分子成像研究。本项目利用活体USPIO加荧光标记TRL4基因敲除小鼠脑缺血后小胶质细胞行MR成像，系统观察针刺干预后12h、24h、48h、72h小胶质细胞在缺血脑区的数量及激活状态，分析针刺调节小胶质细胞时空特征。离体病理分析小胶质细胞以及铁双染色观察梗塞区小胶质细胞数量和激活状态，应用PCR、Wester-blot等定量检测针刺干预时TLR4信号通路变化，查明该变化与USPIO标记小胶质细胞MRI信号变化的相关性，探索和阐明针刺对脑缺血区小胶质细胞激活和相关TLR4信号通路的调控机制，为中医针刺治疗脑缺血炎性反应的机制提供分子影像学依据。

该项课题，在获得过国家自然科学基金委批准之后，就积极开展了相关的研究工作。按照原定计划，严格执行了研究内容和细节，圆满完成各项研究任务，并且获得了良好的成果。已经总结发表SCI论文一篇，另一篇SCI论文已经被接收即将发表。已经发表中文核心期刊论文4篇，另一篇已经接收等待发表。通过本项研究，培养了硕士研究生和博士研究生各一名。. 具体计划的执行情况如下：. 我们及时召开了项目启动会，通过查阅文献，进一步对课题中需要改进和完善的地方进行调整；同时练习造模及尾静脉注射，并开始进行模型制作之后标本的处理，包括取脑、病理染色、基因及蛋白检测、血清学检测等，包括小鼠进行眼眶静脉取血、脑组织取材后固定染色、脑组织实时荧光定量PCR及western blot检测，与此同时加强针刺穴位的定位及针刺操作的练习。 针对MRI成像问题，开始是在联影uMR560-1.5T的MRI设备上调节序列使用小野环形线圈扫描。同时查阅文献撰写相关综述，继续练习造模以提高造模成功率。设置梯度浓度探讨能显示脑梗塞炎性反应的最佳浓度和剂量，同时安排部分课题组成员针对小鼠低氧预适应问题进行相关研究。采用了上海羧菲生物科技有限公司代购的来自加拿大的feraheme进行实验，探索出了该造影剂注射最佳浓度和剂量，由于2018年科室安装了西门子skyra3.0T磁共振机器，配备有大鼠、小鼠专用8通道横向放置线圈，我们重新在西门子机器上调试序列，小鼠脑组织T1WI、T2WI、SWI、DWI横断位及冠状位序列图像质量较联影1.5T有了大大改善。圆满完成了117只TLR4 KO小鼠脑缺血模型相关实验研究。已依据研究指标完成了小鼠的MRI扫描，病灶处基因、蛋白检测及血清指标的检测。在课题进行过程中，完成了课题的中期进展汇报，重点针对研究过程已取得的成果进行了总结，撰写论文，进行及时投稿和发表。