干细胞动员促进血管和神经再生是脑缺血研究的重要方向。Ang-1/Tie-2和整合素α6β1在EPCs动员、血管生成和神经再生中发挥重要作用。干细胞循环与活血化瘀理论具有相似性，其内在相关性目前尚未研究清楚。我们研究发现丹参酮ⅡA与丹皮酚配伍联合rhG-CSF可增加脑缺血大鼠外周血CD34+数量，可能促进血管和神经再生，与Ang-1/Tie-2通路相关，但其作用和整合素α6β1的关系还有待研究。因此提出假说：双丹有效配伍联合rhG-CSF可能是借助Ang-1/Tie-2和整合素α6βl的相互作用，动员EPCs参与脑缺血血管和神经再生；整合素α6β1可能是其关键的分子开关。本项目拟研究两者联合用药对EPCs粘附、迁移、血管生成及脑缺血大鼠血管和神经再生的影响，观察动员效果分析其作用机制和靶点，为阐明干细胞循环与活血化瘀理论的内在相关性提供实验依据，也为干细胞动员治疗脑缺血的临床应用提供新策略。

It is an important research direction of cerebral ischemia that bone marrow stem cells mobilization is beneficial to promote nerve regeneration and angiogenesis. Ang/Tie-2 signaling pathway and integrinα6β1 have been involved in regulating angiogenesis and nerve regeneration, and played important roles in the promotion of endothelial progenitor cells (EPCs) mobilization. In theory, the circulation of stem cell is similar to activating blood circulation to dissipate blood stasis, but its correlations are not clear. Our previous study found that the expression of peripheral blood CD34+ cells were significantly increased by the rhG-CSF joint combination of Tanshinone ⅡA and Paeonol. It could perhaps promote nerve regeneration and angiogenesis, and Ang/Tie-2 signaling pathway has been involved in cerebral ischemia injury in addition. Howerer, further research is needed to observe the relationship and its effects on integrinα6β1.Therefore, we hypothesise that: it is the interaction with Ang/Tie-2 signaling pathway and integrinα6β1 that EPCs mobilization can take part in promoting nerve regeneration and angiogenesis after cerebral ischemia injury treatment by the rhG-CSF joint combination of Tanshinone ⅡA and Paeonol. Integrinα6β1 may be the acting targets. In this study, angiopoiesis, migration, adhesion capacity of EPCs will be examined. Simultaneously we will analyze the molecular mechanism and acting targets by comparing the effect of treatment by combination of Tanshinone ⅡA and Paeonol, rhG-CSF and their combined application on cerebral ischemia injury in rats and observing its mobilization effect on EPCs into blood regulating for angiogenesis and nerve regeneration. The results will help to clarify the theory of the circulation of stem cell correlations with activating blood circulation to dissipate blood stasis, lay the experimental foundation for it, and provide the new prevention methods and new drugs for ischemic cerebrovascular disease in the field of the clinical application of stem cell mobilization at the same time.

干细胞动员促进血管和神经再生是脑缺血研究的重要方向，目前骨髓干细胞动员剂如何在体内实现动员后，向脑缺血损伤区域归巢的靶向性和高效性，预防动员剂的副作用，探寻最佳动员方案等急待解决。本研究分离培养、鉴定大鼠骨髓EPCs，用正交设计优选丹皮酚与丹参酮ⅡA联合rhG-CSF，促进EPCs增殖的最佳剂量配伍依次是6.9969μg/ml、0.8685μg/ml及14.1750ng/ml。丹皮酚对EPCs的存活率有显著性影响（P<0.01）；其次是丹皮酚与丹参酮ⅡA交互作用和丹参酮ⅡA（P<0.05）；随着rhG-CSF剂量的增加，细胞存活率降低，提示丹皮酚和丹参酮ⅡA可能协同rhG-CSF促进EPCs增殖，提高细胞存活率，减少了rhG-CSF的用量，发挥增效减毒的作用，有可能成为安全、高效的干细胞动员剂或者辅助剂。我们前期研究发现双丹方脂溶性组分丹参酮ⅡA与丹皮酚配伍联合rhG-CSF，可增加脑缺血大鼠外周血CD34+数量，可能具有促进血管和神经再生的作用，与Ang-1/Tie-2通路相关。除了通过 Tie2 外，Ang 家族还通过整合素途径介导细胞内信号转导，但其作用与整合素α6β1的关系还有待进一步研究。本实验采用脑室注射重组人血管生长素（rhAngiogenin）上调血管和GoH3抗体封闭整合素a6表达，对比观察EPCs的动员效果及Ang-1/ Tie-2-integrinα6β1通路关键靶点表达的影响。研究发现，与模型组比较，Angiopoietin-1的蛋白表达，GoH3组呈降低趋势，联合+ GoH3组比GoH3组显著增加，且联合组的表达明显高于模型组，表明双丹有效配伍联合rhG-CSF干预，部分抵消了GoH3对Ang-1的抑制作用。与GoH3组比较，联合+ GoH3组外周血CD34+/KDR+阳性细胞数明显增加（p＜0.05），提示其动员作用可能通过激活Integrinα6通路实现。以Brdu+/vWF+、Brdu+/CD34+双染及RECA1检测内皮细胞增殖和血管新生。用Brdu+/DCX+、Brdu+/NeuN+、Brdu+/GFAP+双染确定对大鼠内源性神经再生的影响。研究结果发现双丹有效配伍联合rhG-CSF，可促进脑缺血神经干细胞的增殖分化和血管新生。本研究可为临床上丹皮酚与丹参酮ⅡA联合rhG-CSF动员干细胞，治疗脑缺血疾病提供实验依据。