低氧对共培养体系中内皮细胞eNOS活性及平滑肌细胞增殖与迁移能力的影响

Effects of hypoxia on eNOS activity of endothelial cells and proliferation and migration of smooth muscle cells in a co-culture system

目的探讨在内皮细胞和平滑肌细胞共培养模型中,低氧对内皮细胞中eNOS活性,以及平滑肌细胞增殖和迁移能力的影响。方法利用人肺动脉内皮细胞(HPAEC)与人肺动脉平滑肌细胞(HPASMC)构建共培养模型,经过低氧(3%O_(2))或常氧(20%O_(2))处理后,通过CCK‐8实验、Ki‐67和EdU染色、Western印迹及Transwell小室迁移实验分别检测低氧对HPASMC增殖和迁移的影响;通过试剂盒和Western印迹分别检测低氧对NO释放量和eNOS活性的影响;进一步利用硝普钠(SNP)分解生成NO的特性验证HPAEC释放的NO在调节HPASMC增殖和迁移中的作用。结果低氧能显著促进共培养模型中HPASMC的增殖与迁移能力,并抑制HPAEC中eNOS的活性和NO生成;然而,在HPAEC培养基中添加SNP则能明显逆转由低氧导致的HPASMC增殖与迁移。结论低氧通过抑制共培养体系下HPAEC中eNOS的活性进而减少NO的生成和分泌,NO释放量的减少最终促进了HPASMC的增殖与迁移。

Objective To investigate the effects of hypoxia on eNOS activity in endothelial cells and on the proliferation and migration of smooth muscle cells in a co‐culture model.Methods A co‐culture model of human pulmonary artery endothelial cells(HPAEC)and human pulmonary artery smooth muscle cells(HPASMC)was established.After exposure to hypoxia(3%O_2)or normoxia(20%O_2),the effects of hypoxia on proliferation and migration of HPASMC were examined by CCK‐8 assay,Ki‐67 and EdU staining,Western blotting and Transwell.The effects of hypoxia on NO release and eNOS activity were detected by the testing kit and Western blotting respectively.The role of NO in regulating the proliferation and migration of HPASMC was further verified using sodium nitroprusside(SNP)which could generate NO in the medium.Results Hypoxia could significantly promote the proliferation and migration of HPASMC in the co‐culture model while inhibiting the eNOS activity and NO production in HPAEC.However,supplementation of SNP in HPAECs could significantly reverse the proliferation and migration of HPASMC caused by hypoxia.Conclusion Hypoxia reduces the production and secretion of NO by inhibiting the activity of eNOS in HPAECs in the co‐culture system,and the reduction of NO ultimately promotes the proliferation and migration of HPASMC.