不同间歇低氧暴露内皮细胞与多形核白细胞共培养下核转录因子κB p65表达水平的研究

A Comparative Study of NF-κB p65 Level in Different Intermittent Hypoxia Exposed Co-cultured Endothelial Cells and Polymorphonuclears

目的 探讨不同间歇低氧暴露的内皮细胞和多形核白细胞（ PMN） 共培养后内皮细胞中核转录因子κB p65（ NF-κB p65） 的水平变化。方法 18 只雄性Wistar 大鼠随机分为常氧组和间歇低氧组（ IH 组） 。IH 组暴露于间歇低氧环境中, 氧浓度波动于5. 4% ～20. 7% , 低氧频率为30 次/h,8 h/d,6 周后处死大鼠, 腹主动脉取血, 分离纯化PMN。PMN分别与常氧暴露内皮细胞、IH 暴露内皮细胞（ 低氧频率12 次/h, 共4 h） 共培养4 h。细胞按暴露情况分为常氧内皮＋ 常氧PMN 组, IH 内皮＋常氧PMN组, 常氧内皮＋ IHPMN 组, IH 内皮＋ IHPMN 组。Western blotting 测定内皮细胞中NF-κB p65 浓度, GAPDH 为内参, 以NF-κB p65 /GAPDH 标准化NF-κB p65 的表达量。结果 IH 内皮＋常氧PMN组与常氧内皮＋IHPMN组NF-κB p65 表达量分别为2. 49 ±0. 39 和2. 14 ±0. 33, 两组间差异无统计学意义（ P 〉0. 05） , 但两组均较常氧内皮＋ 常氧PMN 组显著升高（ 0. 68 ±0. 20, P 〈0. 05） 。IH 内皮＋IHPMN组NF-κB p65 表达水平为4. 17 ±1. 48, 较其余三组显著升高（ P 〈 0. 05） 。结论 间歇低氧可分别致内皮细胞和PMN发生炎症反应, 而当两种细胞同时暴露于间歇低氧条件时炎症反应最为严重。上述两种细胞间的相互作用和引发的炎症反应可能是低氧内皮细胞损伤和睡眠呼吸暂停综合征并发心血管合并症的重要原因。

Objective To investigate the expression of nuclear factor kappa B p65 （ NF-κB p65） inendothelial cells when endothelial cells and polymorphonuclears （ PMNs） exposed to different intermittent hypoxia （ IH） and co-cultured. Methods Eighteen male Wistar rats were randomly divided into a normal oxygenic group and an intermittent hypoxia （ IH） group. IH group was exposed to intermittent hypoxia environment as the oxygen concentration fluctuated within the range of 5. 4% -20. 7% , and the frequency of hypoxia was 30 times /h and 8 hours /d. The rats were executed after 6 weeks and blooded from abdominal aorta. PMNs were isolated and purif ed, then incubated with normal oxygenic endothelial cells or IH exposed endothelial cells （ hypoxia frequency was 12 times/h and totally 4 hours） for 4 hours. The cells were divided into 4 groups according to exposing conditions, as normal oxygenic endothelial cells ＋ normal oxygenic PMN group, IH endothelial cells ＋ normal oxygenic PMN group, normal oxygenic endothelial cells ＋ IHPMN group, IH endothelial cells ＋ IHPMN group. Western blotting was applied to measure the NF-κB p65 concentration in endothelial cells. The levels of GAPDH were used as the internal standard. The intensity of NF-κB p65 was normalized to that of GAPDH.Results The level of NF-κB p65 was 2. 49 ±0. 39 and 2. 14 ±0. 33 represented in the IH endothelial cells ＋ normal oxygenic PMN group and normal oxygenic endothelial cells ＋ IHPMN group, without significant difference between two groups （ P 〉 0. 05） , but significantly higher compared with the normal oxygenic endothelial cells ＋ normal oxygenic PMN group （ 0. 68 ±0. 20, P 〈0. 05） . The level of NF-κB p65 was 4. 17 ±1. 48 in the IH endothelial cells ＋ IHPMN group, significantly increased than other three groups （ P 〈 0. 05 ） . Conclusions IH can induce inflammatory reaction in endothelial cells and PMNs, and the degree of inflammation get highest when both cells were exposed to intermittent hypoxia. The interaction between these cells and inflammatory reaction may play an important role in endothelial cells injury and cardiovascular complication in sleep apnea syndrome.