基于血小板微颗粒信使作用研究桃红四物汤对实验性脑缺血血管新生调节机制

Microvascular angiogenesis in ischemia area is an essential link of regulating cerebral ischemia by traditional Chinese medicine which have a therapeutic principle of “Eliminating blood stasis and promoting tissue regeneration”. When under the state of cerebral ischemia, platelets could shed special biological platelets microparticles(PMPs) that contain biological signal factors and , and regulate the function of brain microvascular endothelial cells(BMEC) as messengers . The therapeutic effect of Taohong-siwu decoction (THSW) has been proved in the aspect of treating cerebral ischemia.Our previously research found that THSW could regulate function of platelet and promot angiogenesis. So we want to explore weather THSW can promot angiogenesis in ischemic area via regulate the signal factors carried by PMPs. The aim of this study is to investigate the special regulation by THSW to the biological information carried by PMPs in cerebral ischemia, and study the mechanism of the microvascular angiogenesis by sepcial PMPs. First, rats cerebral ischemia model of rats was established, on which the angiogenesis effects of THSW were observed and the platelet microparticles were extract from plasma to analysis quantity and the transfer information. Second, detect multiplication and vascular endothelial growth factor induction by PMPs in brain microvascular endothelial cells and its downstream transduction pathway, in order to illuminate the mechanism of angiogenesis by adjusting the messenger function of PMPs.

缺血区脑微血管新生是祛瘀生新中药调控脑缺血损伤的重要环节，血小板在脑缺血状态下可以释放携带特异生物因子的血小板微颗粒（PMPs），并以PMPs为信使调节微血管内皮细胞功能，影响脑微血管新生。祛瘀生新经典复方桃红四物汤临床对于缺血性脑中风有明确的作用，前期研究发现其有调节脑缺血大鼠血小板功能和促进血管新生作用。提示PMPs可能为桃红四物汤促进血管形成的作用靶点。本项目旨在探讨桃红四物汤对脑缺血状态PMPs携带生物信息特异性调节和特异性PMPs对脑微血管新生作用机制。通过建立脑缺血模型，考察桃红四物汤对脑缺血损伤保护和血管新生作用，同时提取血浆中血小板微颗粒并考察其数量和携带生物信息分子特征；通过建立脑微血管内皮细胞体外模型，考察桃红四物汤干预的脑缺血PMPs对脑微血管内皮细胞增殖和血管生长因子表达以及传导信号通路影响，明确桃红四物汤通过调节PMPs信使功能促进血管新生的调节机制。

通过血小板微颗粒（PMPs）为信使调控微血管内皮细胞功能，是影响血管新生的一个重要因素。本项目研究表明，桃红四物汤抗脑缺血再灌注损伤，促进脑缺血再灌注损伤后血管生成。桃红四物汤通过降低脑缺血再灌注损伤后氧化应激水平、提高脑源性神经营养因子水平、提高VEGF水平等途径产生抗脑缺血再灌注损伤、促进血管生成等作用。运用蛋白质芯片技术对PMPs颗粒中血管新生蛋白水平进行研究。结果发现，蛋白质芯片上共有79种蛋白，检测出了29种蛋白质的倍数变化大于1.1，11种蛋白变化倍数大于1.2，3种蛋白变化倍数大于1.3。表明大多数这些蛋白质在促进血管生成中具有直接或间接作用。基于倍数变化值，选择Resistin，WISP-1/CCN4，Cyr61/CCN1，Jagged 1和MMP-9差异蛋白进行验证。验证结果表明THSWD高、中组能够显著提高Resistin，WISP-1 / CCN4，Cyr61 / CCN1，Jagged 1和MMP-9蛋白的表达。本项目研究表明THSWD对防止脑缺血损伤有一定的价值，并从血小板微颗粒的角度阐明了THSWD“祛瘀”与“生新络”之间的联系，对于桃红四物汤的开发研究具有一定的参考意义。项目支持发表论文7篇，包括SCI 2篇、中文核心期刊5篇；培养毕业硕士研究生2人，在读硕士研究生1人。