SDF-1/CXCR4轴诱导血管新生在针刺内关改善MCAO大鼠海马微循环中的作用及机制研究

Microcirculation disturbance could impose great impact on the metabolism of the ischemic brain tissue which functional and morphological damages will be more severe accordingly. Acupuncture is an effective means to improve the microcirculation disturbance state in ischemic brain tissue. According to our previous and preliminary research results, it was found that acupuncturing at Neiguan makes a difference to the angiogenesis in the hippocampal CA1 region. It has been confirmed that the SDF-1/CXCR4 axis plays a key role in the induction of angiogenesis. To investigate the effect of angiogenesis induced by SDF-1/CXCR4 axis on the amelioration of microcirculation disturbance state in the region of hippocampal CA1 and the relevant mechanism, the rat MCAO model will be established. The neurobehavioral score will be detected at a different time. HE staining will be performed. The quantity of EPCs and the level of HIF-1 and VEGF among peripheral blood will be detected. The neogenetic microvessel quantity and microcirculation flow in the hippocampal CA1 region will be detected. The expression levels of mRNA and protein of both SDF-1 and CXCR4 will be detected and anlyzed. These effects of acupuncturing at Neiguan will be confirmed by means of the CXCR4 inhibitor AMD3100. The relationship of the factors detected above will be anylazed. Thus, it is anticipated that the effect and relevant mechanism of angiogenesis induced by SDF-1/CXCR4 axis to improve the hippocampal CA1 region microcirculation state resulted from acupuncturing at Neiguan would be revealed, which could provide scientific basis to the therapy of cerebral ischemia by acupuncture.

微循环障碍可影响缺血脑组织代谢，加重脑组织功能和形态结构的损害。针刺是改善缺血脑组织微循环障碍的有效手段。我们的前期研究显示针刺内关穴可增加海马CA1区的血管新生，而SDF-1/CXCR4轴是诱导血管新生的关键机制。为了探索SDF-1/CXCR4轴诱导血管新生在针刺内关穴改善海马CA1区微循环障碍中的作用及机制，本项目拟应用MCAO大鼠模型，在不同时间窗进行神经行为评分，HE染色，外周血中HIF-1、VEGF水平、EPCs数量检测，海马CA1区CD34和CXCR4双阳性新生微血管数量及微循环血流量检测、SDF-1和CXCR4 mRNA及蛋白质表达检测分析，并观察CXCR4抑制剂AMD3100对上述指标的干预作用。通过分析上述指标的变化和相互间的关联及影响，揭示由SDF-1/CXCR4轴诱导的血管新生在针刺内关改善海马CA1区微循环障碍中的作用及机制，为针刺治疗脑缺血性疾病提供科学依据。

本项目主要研究探讨了针刺改善缺血脑组织微循环障碍的相关机制。实验中采用线栓法建立大脑中动脉栓塞（MCAO）大鼠模型，通过神经行为学评分、微循环学、组织形态学、免疫组织化学、实时定量PCR、Western blot、流式细胞仪等方法，观察连续针刺1、3、7、14天进行神经行为评分，HE染色，外周血中HIF-1、VEGF水平、EPCs数量检测，海马CA1区CD34和CXCR4双阳性新生微血管数量及微循环血流量检测、SDF-1和CXCR4 mRN A及蛋白质表达检测分析，并观察CXCR4抑制剂AMD3100对上述指标的干预作用，探讨由SDF-1/CXCR4轴诱导的血管新生在针刺内关改善海马CA1区微循环障碍中的作用及机制。应用SPSS19.0统计学软件进行数据统计及结果分析。.结果显示：1. 针刺内关穴能够显著改善MCAO大鼠神经功能损伤，有效促进其精细运动功能的恢复，其治疗效果与无关对照穴相比有显著性差异。2. 针刺能够显著提高MCAO大鼠外周血中内皮祖细胞数量，显著提高外周血中SDF-1及VEGF的水平，表明针刺内关可动员内皮祖细胞，参与缺血脑组织中微血管的生成。3.针刺内关能够显著提高MCAO大鼠脑微循环血流量以及MCAO大鼠脑组织中SDF-1、CXCR4 mRNA及其蛋白的表达，治疗效果与无关对照穴相比有显著性差异。腹腔注射CXCR4抑制剂AMD3100后可抑制相关mRNA及蛋白的表达，抑制MCAO大鼠神经损伤的恢复及软脑膜微循环血流量及微血管数量。针刺对缺血脑组织中微血管的生成具有调节作用，是其防治脑缺血，促进缺血脑组织血液微循环及神经功能恢复的机制之一。