益气活血法对脑出血后血管新生-胶质细胞活化相互作用的调节机制研究

Angiogenesis and astrocyte (AST) activation are closely related. AST activation can promote angiogenesis by secreting HIF-1α, but if the AST were over activated and glial scar formation, which will be a barrier for angiogenesis. Leukemia inhibitory factor which is secreted from endothelial cells has been shown to inhibit AST activation as a negative feedback signal. So HIF-1α and LIF are the most important factors for the interaction between angiogenesis and AST activation. Our previous work has confirmed that Qi-tonifying and stasis-eliminating therapy can promote angiogenesis after intracerebral hemorrhage (ICH), and domestic researchers shows Qi-tonifying and stasis-eliminating therapy play a vital role in regulating astrocytes activation and inhibiting glial scar formation after ischemia as well. Hence, Qi-tonifying and stasis-eliminating therapy is supposed to regulate the interaction between angiogenesis and AST activation by HIF-1α and LIF, and optimize microvascular reconstruction after ICH. The purpose of this project is to establish the effect of Qi-tonifying and stasis-eliminating therapy on angiogenesis, AST activation, spatial and temporal expression of HIF-1α and LIF, and mutual causality after ICH, to further elucidate its mechanisms in reconstruction of microvascular networks, and to provide more adequate experimental basis for the clinic application.

血管新生与星形胶质细胞（AST）活化密切有关。AST活化通过分泌HIF-1α而促进血管新生过程，若AST过度活化形成胶质瘢痕，则对新生血管起屏障作用；血管内皮细胞通过LIF反馈抑制AST活化，因此HIF-1α/LIF是血管新生-AST活化相互作用的重要枢纽之一。我们前期工作证实益气活血法能促进脑出血后的血管新生，国内同行研究显示益气活血法具有调节脑缺血后星形胶质细胞活化，抑制胶质瘢痕形成的作用。推测益气活血法可能通过HIF-1α/LIF而调节血管新生与AST活化相互作用而优化脑出血后微血管重建过程。本项目拟从血管新生与AST活化相互作用的角度研究益气活血法对脑出血后血管新生、AST活化、HIF-1α/LIF时空表达的影响及其因果关系，进一步揭示益气活血法促进脑出血损伤组织修复的机理，为其临床上促进神经功能恢复提供更充分的实验依据。

背景和目的 血管新生与星形胶质细胞（AST）活化密切有关。AST活化形成胶质瘢痕，对新生血管起屏障作用；HIF-1α和LIF分别是调节血管新生和AST活化的关键因子。本项目拟从血管新生与AST活化相互作用的角度研究益气活血法对脑出血后血管新生、AST活化、HIF-1α/LIF时空表达的影响及其因果关系，进一步揭示益气活血法促进脑出血损伤组织修复的机理。.方法 本研究主要分为3部分。（1）立体定位向脑内苍白球注入大鼠自体动脉血(100 µL)复制脑出血模型。采用益气活血法经典方—补阳还五汤灌胃进行干预；（2）造模后选用HIF-1α抑制剂2-methoxyestradiol（2ME2，5mg/kg) 腹腔注射；（3）造模后选用LIF（100ng）直接注入苍白球。免疫组化方法检测脑出血损伤区BrdU+/vWF+标记新生血管和BrdU+/GFAP+标记增殖的星形胶质细胞，免疫组化检测GFAP、HIF-1α、VEGF、LIF和LIFR的时空分布。荧光定量RT-PCR和Western bot检测GFAP、HIF-1α、VEGF、LIF和LIFR表达变化。.结果 脑出血后3天血肿周围即可以见到BrdU+/vWF+和BrdU+/GFAP+标记的细胞核，且持续增高至14天。脑出血后血肿周围可见星形胶质细胞胞体肥大、并相互缠绕形成胶质瘢痕。同时免疫组化检测在血肿周围可见大量HIF-1α、VEGF、LIF和LIFR阳性表达；HIF-1α和VEGF表达于血管内皮细胞，而LIF和LIFR表达于星形胶质细胞；脑出血后3天GFAP、HIF-1α、VEGF、LIF和LIFR表达也较假手术组明显增高。HIF-1α表达从7天开始明显降低，VEGF表达持续增高至21天，GFAP、LIF和LIFR蛋白表达增高至14天。在采用补阳还五汤干预后，BrdU+/vWF+标记的细胞核明显增多，但是BrdU+/GFAP+标记的细胞核明显减少，而且补阳还五汤能促进HIF-1α和VEGF的表达，抑制GFAP、LIF和LIFR的表达。在采用HIF-1α抑制剂2ME2干预后，BrdU+/vWF+标记的细胞核明显减少，但是BrdU+/GFAP+标记的细胞核明显增多，GFAP、LIF和LIFR的表达明显升高。在采用外源性LIF干预后，BrdU+/GFAP+标记的细胞核明显增多，GFAP和LIFR的表达明显上调，BrdU+/vWF