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在低氧条件下,观察大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells, PASMCs)中活性氧(reactive oxygenspecies, ROS)的变化,探讨 ROS 的变化是否通过调控低氧诱导因子 -1α (hypoxia-inducible factor 1α, HIF-1α)的表达影响 PASMCs的增殖。采用组织块法原代培养大鼠PASMCs,分成3 组:常氧组(21% O2, 24 h),低氧组(5% O2, 24 h),低氧+ Mn-TBAP组(5% O2,24 h,Mn-TBAP 是一种ROS 清除剂)。用激光共聚焦显微镜荧光染色法检测细胞内ROS 的变化;用RT-PCR 和免疫组织化学方法分别测定HIF-1α mRNA 和蛋白的表达;用MTT 法检测细胞增殖程度。结果显示:(1) 低氧组 PASMCs内ROS水平明显高于常氧组(P<0.05),低氧+ Mn-TBAP 组ROS 水平明显低于低氧组(P<0.05),但仍高于常氧组(P<0.05) ;(2)低氧组及低氧+ Mn-TBAP 组的HIF-1α mRNA 和蛋白表达均高于常氧组(P<0.05),且低氧组表达高于低氧+ Mn-TBAP 组(P<0.05) ;(3) 低氧组细胞增殖明显高于常氧组和低氧+ Mn-TBAP 组(P<0.05),低氧+ Mn-TBAP 组细胞增殖高于常氧组(P<0.05)。结果表明:在低氧条件下大鼠PASMCs 中 ROS 水平明显升高,且ROS 的变化能够调节HIF-1α的表达,进而影响平滑肌细胞的增殖,提示ROS 可能在肺动脉高压的发病机制和低氧信号转导中具有重要作用。
To observe the changes of reactive oxygen species (ROS) in rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions and investigate whether ROS changes through the regulation of hypoxia-inducible factor 1α, HIF-1α) affect the proliferation of PASMCs. PASMCs were primarily cultured by tissue block method and divided into 3 groups: normoxia group (21% O2, 24 h), hypoxia group (5% O2, 24 h) , 24 h, Mn-TBAP is an ROS scavenger). The changes of intracellular ROS were detected by laser scanning confocal microscopy. The expression of HIF-1α mRNA and protein were detected by RT-PCR and immunohistochemistry. The proliferation of cells was detected by MTT assay. The results showed that: (1) ROS levels in PASMCs in hypoxia group were significantly higher than those in normoxia group (P <0.05); ROS levels in hypoxia + Mn-TBAP group were significantly lower than those in hypoxia group (P <0.05) (2) The expression of HIF-1αmRNA and protein in hypoxia group and hypoxia + Mn-TBAP group were higher than that in normoxia group (P <0.05), and the expression of HIF-1α in hypoxia group was higher than that in normoxia group (P <0.05); (3) The cell proliferation in hypoxia group was significantly higher than that in normoxia group and hypoxia + Mn-TBAP group (P <0.05) Higher than normoxic group (P <0.05). The results showed that the level of ROS in PASMCs was significantly increased under hypoxic conditions, and the changes of ROS could regulate the expression of HIF-1α, and then affect the proliferation of smooth muscle cells, suggesting that ROS may play an important role in the pathogenesis of pulmonary hypertension and hypoxia signaling Transduction has an important role.