疮灵液载入胶原对血管新生影响的实验研究
The Effects of Chuangliye decoction loading into modified collagen matrices on angiogenesis
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摘要: 目的 探讨疮灵液及其载入胶原后对血管新生的影响。方法 将不同剂量的疮灵液及其载入胶原后，通过鸡胚尿囊膜模型（CAM）测定筛选其血管新生疗效；通过血管内皮细胞增殖实验（MTT）、迁移划痕实验、血管生成素1(Ang1)、血管内皮生长因子(VEGF)与整合素(Integraаv)Western blot检测探查筛选适合剂量的疮灵液胶原的作用靶点与机制。结果 CAM实验显示疮灵液0.2 mL及0.3 mL组血管新生少于控制组（P<0.05），创灵液0.2 mL与0.3 mL组之间没有明显差异；载入胶原后其抑制微血管新生作用得到逆转，结果与控制组无统计学差异。选择疮灵液0.2 mL及其载入胶原进行细胞实验，结果显示修饰后胶原可以显著促进HUVEC的增殖，增殖率达86%，而单纯疮灵液则抑制了HUVEC增殖约21%，相同剂量疮灵液载入胶原后，增殖率得到升高，高于控制组约24%（P<0.01）；疮灵液组显著抑制了细胞的迁移运动，在12、24 h时均显著低于其他各组；疮灵液载入胶原后，细胞迁移速度开始加速，24 h迁移速度显著快于控制组（P<0.01）；机制在于疮灵液组Integraаv蛋白表达明显低于控制组，而Ang1表达高于控制组，载入胶原后Integraаv、VEG F及Ang1蛋白表达均明显上升。结论 疮灵液抗血管内皮细胞的迁移与增殖，抑制血管新生；将疮灵液载入胶原后，可以改善其抑制血管新生的作用，有利血管成熟，提高了其促进创面愈合的疗效。Abstract: OBJECTIVE To investigate the effects of Chuangling liquid and its loaded Collagen on angiogenesis.METHODS Different dosages of Chuangling liquid and its collagen loaded liquid were detected for through the chicken embryo allantois membrane model(CAM) by the optimal effects of angiogenesis. The vascular endothelial cell proliferation experiment (MTT)， the migration assay and the protein expression angiogenin1 (Ang1)， vascular endothelial growth factor (VEGF) and integrin(Integraav) with Western blot method were used for the detection of mechanism.RESULTS The CAM experiments showed that the angiogensis in 0.2 mL and 0.3 mL Chuangling liquid groups were significantly less than that of control group(P<0.05)， while there was no significant difference between 0.2 mL and 0.3 mL Chuangling liquid group. This antiangiogenic effects were reversed by the same dosage of Chuangling liquid loading into collagen with their effects reaching to the effects of control group. The 0.2 mL dosage of Chuangling liquid loading into collagen and/or loading into the collagen were chosen for the further experiment. The HUVEC proliferation rate of control group increased 86%， as compared with the group of Chuangling liquidwhich decreased. After loading into collagen， the proliferation rate was increased significantly higher than the results of control group with about 24%(P<0.01). The cell migration movement of 12hours， 24hours in the group of Chuangling liquidwere significantly lower than those in other group. When loading into collagen， cell migration rate began to accelerate and the migration rate significantly faster than that of control group on 24hours(P<0.01); Mechanism detection showed that the protein expressions of Chuangling liquidwith Integraаv was significantly lower than the results of control group， and the higher of Ang1 expression. After loading into collagen， the protein expressions of Integraаv， VEGF and Ang1 were increased significantly.CONCLUSION The study showed that negative effects on vascular endothelial cell migration and proliferation and thus inhibiting angiogenesis. Chuangling liquid loaded into Collagen may reduce the inhibition to angiogensis and be beneficial for blood vessels formation， in order to improve wound healing.
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