Alg-g-PEI/pVEGF复合物体内促血管生成作用

Angiogenic activity of alginate-graft-PEI/pVEGF complexes in vivo

摘要:

旨在研究氧化海藻酸——低分子量聚乙烯亚胺两性刷型共聚物 (Algin-a-te-graft-PEI, Alg-g-PEI) 与血管内皮生长因子质粒 (pVEGF) 复合后对体内血管生成的影响。采用细胞和斑马鱼实验检测了Alg-g-PEI/pVEGF复合物的毒性；通过凝胶电泳实验评价了Alg-g-PEI对DNA的保护作用；利用鸡胚绒毛尿囊膜 (CAM) 和斑马鱼模型，选用PEI 25K/pVEGF作为阳性对照、生理盐水为空白对照，观察复合物对血管新生的促进作用。结果发现：Alg-g-PEI对质粒有较好的保护作用；Alg-g-PEI/pVEGF复合物具有较低的细胞、斑马鱼毒性，能显著促进CAM和斑马鱼的血管生成，且具有剂量依赖性，当pVEGF用量等于2.4 μg/CAM时，复合物对CAM血管生成的促进作用最明显，血管面积和CAM面积比 (VA/CAM) 为44.04％，高于阳性对照组 (35.90％) 和空白对照组 (24.03％) (**P＜0.01)。复合物对斑马鱼血管新生的促进作用随氮磷比 (N/P) 的增加而增强，其中N/P=110时，血管新生最明显：肠下血管总长度和面积分别为1.11 mm和1.70×103像素，高于空白对照组 (0.69 mm和0.95×103像素) (**P＜0.01) 和阳性对照组 (0.82 mm和1.11×103像素) (**P＜0.01) 。综上所述，Alg-g-PEI/pVEGF能于体内促进血管生成，该载体有望用于临床缺血性疾病的治疗。

Abstract:

To study the angiogenic activity of amphoteric brush-type copolymer complex of alginate-graft-PEI/pVEGF (Alg-g-PEI/pVEGF) in vivo, we evaluated the toxicity of Alg-g-PEI/pVEGF complexes to rMSCs and zebra fish first. Then, we used gel retardation assay to investigate the protection of complex to pDNA against DNase I, serum and heparin. For in vivo study, we evaluated the angiogenic activity of Alg-g-PEI/pVEGF complexes by using CAM and zebra fish as animal models, PEI 25K/pVEGF and saline as positive and negative controls. Our results show that Alg-g-PEI protected pVEGF from enzymolysis and displacement of heparin in some degree, and its complexes with pVEGF were less toxic to rMSCs and zebra fish. Alg-g-PEI/pVEGF complexes induced significant angiogenesis, which was dosage-dependent. In CAM, when the dosage of pVEGF was 2.4 μg/CAM, Alg-g-PEI group achieved the maximum of angiogenesis, and the area ratio of vessel to the total surface was 44.04%, which is higher than PEI 25K group (35.90%) and saline group (24.03%) (**P<0.01). In zebra fish, the angiogenesis increased with the increase of N/P ratios of Alg-g-PEI/pVEGF complexes in our studied range; when N/P ratio was 110, the optimal angiogenesis was obtained with vessel length of 1.11 mm and area of 1.70×103 pixels, which is higher than saline group (0.69 mm and 0.94×103 pixels) (**P<0.01) and PEI 25k group (0.82 mm and 1.11×103 pixels) (**P<0.01). Our results demonstrate that Alg-g-PEI/pVEGF significantly induces angiogenesis in CAM and zebra fish, and has a great potential in therapeutic angiogenesis.