复方通络饮干预Shh信号介导内皮祖细胞参与脑梗死后血管新生机制研究

Angiogenesis plays an important role in cerebral ischemia injury repairment, recent studys have shown that Sonic hedgehog signal could mediate endothelial progenitor cells (EPCs) to participate in the angiogenesis after ischemic stroke. Fufangtongluo decoction has the function of nourishing kidney and liver, also eliminating phlegm and freeing channels, which was derived from traditional theory for the treatment of stroke from professor Zhongying Zhou. Fufangtongluo decoction has clinical curative effect obviously. Previous researches have confirmed that the drug can protect endothelial cell, up-regulate the protein expression of Shh and promote angiogenesis, but the mechanism is unknown. Combined with kidney advocate bone, brain for medullary sea theory of traditional Chinese medicine, We hypothesis that the drug through intervening Shh expression and mediating bone Endothelial progenitor cells(EPCs) mobilization and homing to ischemic zone, then formation to new blood vessels. In order to verify the hypothesis, we will employ flow cytometry, immunofluorescence technique, confocal laser and Laser speckle imaging techniques , explore the possible mechanism of angiogenesis from cell culture and in vivo levels, clarify the mechanism of the drug through nourishing kidney and liver, producing brain marrow, provide more theoretical basis for the application of the Fufangtongluo decoction.

血管新生在脑缺血损伤修复中起重要作用。最近研究表明Shh可介导内皮祖细胞（EPCs）参与脑梗死后血管新生。复方通络饮为总结国医大师周仲瑛教授治疗脑梗死经验基础上遣方而成，有“滋肾养肝，化痰消瘀”之效，临床研究证实该方可改善脑梗死患者神经功能评分，减轻血管内皮细胞损伤；实验研究表明该方可上调脑梗死大鼠Shh表达，促进血管新生，缩小脑梗死体积，但机制不详。根据中医“肾主骨生髓，脑为髓海”理论，结合该方通过“补益肝肾”而“化瘀生新”的治疗特点，我们提出脑梗死后该方是否通过干预Shh表达影响骨髓EPCs动员及归巢，进而增殖分化为新生血管。为了验证这一推断，本研究将应用大鼠永久性大脑中动脉梗死模型，通过流式细胞、免疫荧光、激光散斑成像等技术从体内到细胞培养不同层面探讨该方影响EPCs参与血管新生的可能机制，以期阐明该方通过“补肝肾、化痰瘀”而“生脑髓”的作用机理，为该方的应用开发提供更多的理论依据。

血管新生在脑缺血损伤修复中起重要作用。最近研究表明Shh可介导内皮祖细胞（EPCs）参与脑梗死后血管新生。复方通络饮为总结国医大师周仲瑛教授治疗脑梗死经验基础上遣方而成，有“滋肾养肝，化痰消瘀”之效，临床研究证实该方可改善脑梗死患者神经功能评分，保护血管内皮细胞功能；实验研究表明该方可上调脑梗死大鼠Shh表达，促进血管新生，缩小脑梗死体积；然而其保护机制不详。根据中医“肾主骨生髓，脑为髓海”理论，结合该方通过“补益肝肾”从而“化瘀生新”的治疗特点，我们通过建立大鼠永久性大脑中动脉梗死模型（pMCAO）,通过流式细胞、免疫荧光、激光散斑成像等技术从体内到细胞培养等不同层面探讨了该方影响EPCs参与血管新生的可能机制。研究结果明确了复方通络饮能够动员pMCAO大鼠骨髓EPCs向脑梗死区域定向归巢；证实了复方通络饮能够增加缺血侧脑血流灌注，促进血管新生作用；初步确定了复方通络饮通过激活Shh信号通路诱导内皮祖细胞定向迁移、归巢，促进脑梗死后血管新生的作用机制，阐明了该方通过“补肝肾”而“生脑髓”的作用机理。基于以上研究结果，复方通络饮在脑梗死治疗中的临床应用先后推广应用于江苏省中西医结合医院，江苏省第二中医院及南京市中西医结合医院等三级甲等中医院，并获得江苏省医学新技术引进二等奖，南京中医药科学技术三等奖。本研究结果为该方的下一步应用开发提供更多依据。