复合促血管再生因子/葡聚糖/PLGA纳米微球纤维蛋白胶的血管再生鸡尾酒疗法研究

In our previous study, we found G-CSF and EPO released from fibrin gel could mobilize stem cells and progenitor cells, and inhibit the apoptosis of skeletal muscle to promote neovascularization. Based on this study, the angiogenesis effect of VEGF, arteriogenesis effect of FGF-2 and control-releasing function of PEG/PLGA nano-microspheres, to overcome the deficiency of the single vasculogenic cytokine and its short half-life, a new neovascularizational treatment of intramuscular injection of PEG/PLGA nano-microspheres fibrin gel carried compound vasculogenic cytokine cocktail (including VEGF, FGF-2, EPO and G-CSF) is proposed. The objective is to study the coating rate，in vitro and in vivo control-releasing effect of the PEG/PLGA nano-microspheres fibrin gel carried compound vasculogenic cytokines; to explore the synergistic vasculogenic mechanisms of compound cytokines in the aspects of mobilization and activation of stem cells, progenitor cells and inflammatory cells, activation of arterial endothelial cells, density of capillary, small artery and collateral artery, and cell apoptosis; to explore the content ratio of compound cytokines that can result in the best neovascularizational effect especially arteriogenesis effect. The significance of this study is to provide a new idea and treatment for ischemic vascular disease.

在前期研究发现携载促红细胞生成素EPO和粒细胞集落刺激因子G-CSF纤维蛋白胶可以动员干祖细胞和抑制骨骼肌凋亡来促血管再生的基础上，依据血管内皮生长因子VEGF的血管生成作用、成纤维细胞生长因子FGF-2的动脉生成作用和葡聚糖/聚乳酸乙醇酸PLGA纳米微球的载药缓释功能，以克服了单一细胞因子血管再生作用的不足及其半衰期短的缺点，提出肌注复合促血管再生因子(VEGF、FGF-2、EPO和G-CSF)/葡聚糖/PLGA纳米微球纤维蛋白胶的血管再生鸡尾酒疗法。探讨其包封率、体外和体内缓释效果；在干祖细胞、炎性细胞动员活化，动脉内皮细胞的活化，毛细血管密度，小动脉密度，侧支动脉密度，细胞凋亡等方面复合促血管再生因子的协同促血管再生作用机制；获得最佳血管再生特别是侧支动脉生成作用的复合血管再生因子含量比例。从而为缺血性血管疾病的血管再生治疗研究提供一种新思路和新方法。

以血管生长因子为基础的治疗性血管再生治疗有很多缺陷，例如血管再生过程是一个复杂的过程，需要多个血管再生因子的参与，血管再生因子的半衰期很短。因此，我们将粒细胞集落刺激因子（G-CSF）、促红细胞生成素（EPO）和血管内皮生长因子（VEGF）联合葡聚糖/聚乳酸-乙醇酸（PLGA）缓释微球来进行治疗性血管再生研究。制备了含有G-CSF、EPO或VEGF的葡聚糖微球，并将其封装在PLGA微球中，得到了复合血管再生因子/葡聚糖/PLGA微球。研究其基本特性。将复合血管再生因子/葡聚糖/PLGA微球注入大鼠缺血下肢，检测缺血肌组织的血管再生。微球体外释放血管再生因子达 4周以上。结合VEGF、EPO和G-CSF等血管再生因子的血管再生治疗可促进B细胞淋巴瘤-2和基质细胞衍生因子1的表达、增殖细胞核抗原和c-x-c趋化因子受体4阳性细胞的动员。VEGF、EPO和G-CSF等血管再生因子联合治疗组的毛细血管密度和平滑肌α-肌动蛋白阳性血管密度高于单因素治疗组。与单因素治疗相比，使用葡聚糖/PLGA微球联合持续缓释VEGF、EPO和G-CSF等血管再生因子具有更显著的促血管再生作用。这种联合治疗可能是缺血性血管疾病的一种有前途的治疗方法。