乌司他丁调节GSK-3β/β-catenin信号通路对人主动脉夹层血管平滑肌细胞增殖、迁移及凋亡的影响

    Effect of ulinastatin on proliferation, migration and apoptosis of vascular smooth muscle cells in human aortic dissection by regulating GSK-3β/β-catenin signaling pathway

      摘要
    • 摘要:
      目的 探讨乌司他丁（ulinastatin，UTI）调节糖原合成酶激酶-3β（glycogen synthase kinase-3β，GSK-3β）/β-连环蛋白（β-catenin）信号通路对人主动脉夹层（aortic dissection，AD）血管平滑肌细胞（vascular smooth muscle cell，VSMC）增殖、迁移及凋亡的影响。
      方法 将AD患者切除的血管用组织块贴壁法进行原代VSMC提取，并传代培养；且进行考马斯亮蓝染色及α-SMA免疫荧光染色鉴定细胞。体外培养的VSMC随机分为Control组、L-UTI组（500 μmol/L UTI）、H-UTI组（1 500 μmol/L UTI）、H-UTI+pcDNA-NC组（1 500 μmol/L UTI+转染pcDNA-NC）、H-UTI+pcDNA-GSK-3β组（1 500 μmol/L UTI+转染pcDNA-GSK-3β）。qRT-PCR检测各组细胞中GSK-3β相对表达水平；CCK8及平板克隆形成实验测定各组细胞的增殖；划痕实验观察各组细胞的迁移；流式细胞仪测定各组细胞的凋亡；Western blot测定各组细胞中增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）、B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）、GSK-3β/S1P/S1PR3信号通路相关蛋白（p-GSK-3β/GSK-3β、β-catenin）的表达。
      结果 与Control组相比，L-UTI组、H-UTI组细胞的存活率、克隆数、划痕愈合率、PCNA、Bcl-2、p-GSK-3β/GSK-3β、β-catenin蛋白表达降低，而细胞凋亡率、Bax蛋白表达升高（P<0.05，P<0.01）；与H-UTI组或H-UTI+pcDNA-NC组相比，H-UTI+pcDNA-GSK-3β组细胞的存活率、克隆数、划痕愈合率、PCNA、Bcl-2、p-GSK-3β/GSK-3β、β-catenin蛋白表达升高，而细胞凋亡率、Bax蛋白表达降低（均P<0.05）。
      结论 UTI可能是通过调节GSK-3β/β-catenin信号通路，诱导AD患者VSMC凋亡，抑制细胞增殖、迁移。

       

      Abstract:
      AIM To investigate the effect of ulinastatin (UTI) on the proliferation, migration and apoptosis of vascular smooth muscle cells (VSMCs) in human aortic dissection (AD) by regulating the glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway.
      METHODS Primary VSMCs were extracted from the excised blood vessels of AD patients using tissue block adhesion method and were passaged for culture. Coomassie Brilliant Blue staining and α-SMA immunofluorescence staining were performed to identify cells. VSMCs cultured in vitro were randomly grouped into Control group, L-UTI group (500 μmol/L UTI), H-UTI group (1500 μmol/L UTI), H-UTI+pcDNA-NC group (1500 μmol/L UTI+transfected with pcDNA-NC) and H-UTI+pcDNA-SK-3β group (1500 μmol/L UTI+transfected with pcDNA-SK-3β). QRT-PCR was applied to detect the relative expression level of GSK-3β of cells in each group, CCK8 and plate clone formation experiments were performed to detect the proliferation of cells in each group, scratch experiment was conducted to detect the migration of cells in each group, flow cytometry was applied to detect apoptosis of cells in various groups and Western blot was used to detect the expression of proliferating cell nuclear antigen (PCNA), B cell-lymphotumor-2 (Bcl-2), Bcl-2 associated X protein (Bax), and GSK-3β/S1P/S1PR3 signaling pathway related proteins (p-GSK-3β/GSK-3β, β-catenin) of cells in each group.
      RESULTS Compared with those in Control group, the survival rate, clone number, scratch healing rate, PCNA, Bcl-2, p-GSK-3β/GSK-3β and β-catenin protein expressions were lower in L-UTI group and H-UTI group, while the apoptosis rate and Bax protein expression were higher (P<0.05, P<0.01). Compared with those in H-UTI group or H-UTI+pcDNA-NC group, the survival rate, clone number, scratch healing rate, PCNA, Bcl-2, p-GSK-3β/GSK-3β and β-catenin protein expressions of cells were higher in H-UTI+pcDNA-SK-3β group, while the apoptosis rate and Bax protein expression were lower (all P<0.05).
      CONCLUSION UTI may induce VSMC apoptosis and inhibit cell proliferation and migration in AD patients by regulating the GSK-3β/β-catenin signaling pathway.

       

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