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目的揭示补阳还五汤联合骨髓间充质干细胞(MSCs)移植促进脑缺血血管新生的机制。方法选取雄性SD大鼠96只,随机分为脑缺血再灌注模型组、内皮细胞抑制素组、MSCs移植组及补阳还五汤联合MSCs移植组。参照改良的Longa法建立大鼠脑缺血再灌注模型(MCAO)。模型组在立体定位仪下,用微量进样器通过侧脑室给予生理盐水10μl;抑制素组给予内皮细胞抑制素阿瓦斯汀5μl,生理盐水5μl;MSCs组及联合组给予阿瓦斯汀5μl、培养的MSCs 5μl。联合组术前3天给予补阳还五汤(10ml/kg)灌胃,其它各组以同等体积的生理盐水灌胃,每日一次。模型组、抑制素组及MSCs组48 h后,采集实验动物血清和脑组织;联合组分别于12h、24h、36h、48h采集,采用免疫组化法检测大鼠血清VEGF及缺血脑组织VEGF的表达;应用免疫印迹蛋白法检测VEGFR2、p-VEGFR2、ERK、p-ERK、AKT、p-AKT的蛋白含量。结果抑制素组与模型组相比,大鼠血清及脑组织VEGF含量明显降低(P<0.05);ERK、AKT含量变化不大(P>0.05);VEGFR2、p VEGFR2、p ERK和p AKT表达均显著下降(P<0.05)。MSCs组与抑制素组相比,大鼠血清及脑组织VEGF含量明显升高(P<0.05);ERK、AKT含量影响不大(P>0.05);VEGFR2、p-VEGFR2、p-ERK和p-AKT表达均升高(P<0.05);联合组与抑制剂组相比,除ERK外,所有指标均有升高,以24h、36h、48h三个时间点最为明显(P<0.05、P<0.01)。结论内皮细胞抑制素(阿瓦斯汀)可通过抑制VEGFR2及其磷酸化,降低ERK、AKT的磷酸化水平,阻止经MAPK途径的胞內信号转导途径,从而抑制内皮细胞的增殖和迁移,阻止新生血管的形成;MSCs组及联合组主要经VEGFR-2所介导的信号通路及PI3K/AKT信号途径,诱导内皮细胞增殖和迁移,促进血管新生,对ERK途径的胞內信号影响不大。
Objective To reveal the mechanism of Buyang Huanwu Decoction combined with bone marrow mesenchymal stem cells (MSCs) transplantation to promote cerebral ischemia angiogenesis. Methods 96 male SD rats were randomly divided into cerebral ischemia-reperfusion model group, endostatin group, MSCs transplantation group and Buyang Huanwu Decoction combined MSCs transplantation group. A rat model of cerebral ischemia-reperfusion (MCAO) was established according to a modified Longa method. Under stereotaxic instrument, the model group was given 10μl normal saline through lateral ventricle with microinjector; 5μl endostatin Avastin and 5μl normal saline were given in the statin group; 5μl Avastin was given to the MSCs group and the combination group, and cultured Of MSCs 5μl. The combination group was administered with Buyang Huanwu Decoction (10ml / kg) orally 3 days before operation, and the other groups were given gavage with the same volume of normal saline once daily. The model group, the inhibin group and the MSCs group were collected after 48 h, serum and brain tissue were collected; the combination group were collected at 12h, 24h, 36h, 48h, immunohistochemical method was used to detect serum VEGF and ischemic brain tissue VEGF The protein levels of VEGFR2, p-VEGFR2, ERK, p-ERK, AKT and p-AKT were detected by Western blotting. Results Compared with model group, the content of VEGF in serum and brain tissue of rats decreased significantly (P <0.05); the content of ERK and AKT did not change much (P> 0.05); the expression of VEGFR2, p VEGFR2, p ERK and p AKT Decreased significantly (P <0.05). Compared with the statin group, the levels of VEGF in serum and brain tissue of MSCs group were significantly increased (P <0.05); the contents of ERK and AKT were not significantly affected (P> 0.05); VEGFR2, p-VEGFR2, (P <0.05). Compared with the inhibitor group, all the indexes in the combined group except ERK increased at the three time points of 24h, 36h and 48h (P <0.05, P <0.01). Conclusions Endostatin (Avastin) can inhibit the proliferation and migration of endothelial cells by inhibiting VEGFR2 and its phosphorylation, reducing the phosphorylation of ERK and AKT, and preventing the intracellular signal transduction via MAPK pathway The formation of neovascularization. The MSCs group and the combination group mainly induced by VEGFR-2 and PI3K / AKT signaling pathway, which induced the proliferation and migration of endothelial cells and promoted the angiogenesis and had little effect on the intracellular signal of ERK pathway.