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    过表达vWF对人脐静脉内皮细胞生物学影响的实验研究

    Biological effects of overexpression of vWF on human umbilical vein endothelial cells

      摘要
    • 摘要:
      目的 探讨过表达血管性血友病因子(vWF)对人脐静脉内皮细胞(HUVECs)增殖、迁移、侵袭、凋亡及成管能力的影响。
      方法 设计构建5种过表达vWF的质粒CRISPRa载体，将HUVECs分为实验组(5组)与对照组。将5种质粒分别转染实验组细胞，Western blotting检测各组细胞vWF的表达效果，选取优势组。通过细胞增殖实验、细胞划痕实验、细胞侵袭实验、细胞凋亡实验、细胞成管实验等，观察过表达vWF对HUVECs增殖、迁移、侵袭、凋亡及成管能力的影响。Western blotting检测5个常见通路蛋白因子在2组细胞中的表达，探测可能相关的机制。
      结果 成功转染后各组HUVECs的vWF蛋白表达水平均明显升高，与对照组比较差异有统计学意义(P < 0.05)。其中vWF-OE4组过表达量最高，故选取该引物序列进行后续实验。与对照组相比，vWF-OE组细胞增殖减慢，细胞划痕48 h迁移率降低、Transwell细胞侵袭率降低(P < 0.05)，细胞凋亡率、血管形成能力、HUVECs生成小管的长度均增高(P < 0.05)，JUN、P53蛋白含量均增高(P < 0.05)。
      结论 过表达vWF对HUVECs增殖、迁移、侵袭行为具有显著抑制作用，促进凋亡，成管能力增强。

       

      Abstract:
      Objective To investigate the effects of overexpression of von Willebrand factor (vWF) on the proliferation, migration, invasion, apoptosis, and tube formation of human umbilical vein endothelial cells (HUVECs).
      Methods Five vWF-overexpressing plasmid CRISPRa vectors were designed and constructed, and HUVECs were divided into the experimental group (5 groups) and control group.Five plasmids were transfected into the experimental group, and the expression effect of vWF in each group was detected by Western blotting, and the dominant group was selected.The effects of vWF overexpression on the proliferation, migration, invasion, apoptosis and tube formation of HUVECs were observed by cell proliferation assay, scratch assay, cell invasion assay, apoptosis assay, and cell tube formation assay.Western blotting was used to detect the expression of 5 common pathway protein factors in the two groups of cells and explore the possible related mechanisms.
      Results Western blotting showed that the expression level of vWF in HUVECs after successful transfection was significantly higher than that in the control group (P < 0.05).Among them, the overexpression of vWF in the vWF-OE4 group was significantly higher than that of other groups, so the primer sequence was selected for subsequent experiments.Compared with the control group, the cell proliferation in vWF-OE group was slowed down, the cell scratch 48 h mobility was decreased, the Transwell cell invasion rate was decreased (P < 0.05);the apoptosis rate, angiogenesis ability and the length of HUVECs producing tubules were increased (P < 0.05);the protein contents of JUN and P53 were increased (P < 0.05).
      Conclusions Overexpression of vWF can significantly inhibit the proliferation, migration and invasion of HUVECs, promote apoptosis and enhance the ability of tube formation.

       

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