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c—src／NADPH氧化酶1／活性氧对中性粒细胞弹力蛋白酶诱导气道黏蛋白5AC表达的影响 被引量：1

Effects of c-src/Nox1/ROS on mucin5AC expression induced by neutrophil elastase in airway epithelial cells
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摘要 目的探讨中性粒细胞弹力蛋白酶（NE）诱导气道黏液高分泌的上游信号调节机制。方法体外培养人气道BEAS-2B上皮细胞，将细胞分为空白对照组（不加任何刺激）、NE组（NE刺激）、NE＋PP2组（NE刺激合并c—src抑制剂PP2）、NE＋c—src siRNA组（NE刺激合并c—src siRNA）、NE＋Nox1 siRNA组[NE刺激合并NADPH氧化酶1（Nox1）siRNA]、阴性siRNA对照组（加入阴性对照siRNA）及NE＋阴性siRNA组（NE刺激合并阴性对照siRNA）为干预条件。检测干预前后各组细胞中活性氧含量、Nox1蛋白含量、黏蛋白5AC的蛋白及mRNA水平。用四甲基偶氮唑盐法测定各组细胞活性；活性氧试剂盒测定活性氧相对含量；逆转录PCR法检测各组黏蛋白5AC mRNA水平；ELISA法分析细胞黏蛋白5AC的蛋白相对含量；Western blot法检测Nox1蛋白及c—src蛋白的相对含量。结果NE组细胞中Nox1蛋白相对含量为0．88±0．12，高于空白对照组的0．32±0．09（t=9．12，P=0．003）；活性氧相对含量为0．76±-0．09，高于空白对照组的0．18±0．02（t=9．44，P=0．003）；黏蛋白5AC蛋白相对含量为0．82±0．09，基因转录水平为0．77±0．05，均高于空白对照组（分别为0．21±0．11和0．18±0．08，t值分别为7．75和6．13，P值分别为0．004和0．006）。NE＋c—src siRNA组细胞的Nox1蛋白相对含量（0．39±0．08）、活性氧相对含量（0．29±0．05）均低于NE组（t值分别为5．43和5．60，均P=0．007）；黏蛋白5AC蛋白相对含量（0．38±0．09）及mRNA水平（0．41±0．04）低于NE组（t值分别为5．28和4．09，P值分别为0．008和0．034）。NE＋PP2组活性氧相对含量为0．41±0．11，Nox1蛋白相对含量为0．44±0．05，黏蛋白5AC蛋白及mRNA水平分别为0．48±0．08和0．46±0．07，均低于NE组（均P〈0．05）。NE＋Nox1 siRNA组中活性氧相对含量（0．19±0．06）、黏蛋白5AC蛋白相对含量（0．31±0．05）及mRNA水平（0．32±0．06）也低于NE组（均P〈0．05）。结论c—src／Nox1参与了，NE诱导的活性氧活化，是气道上皮细胞黏蛋白5AC合成及分泌的上游信号调节因子。 Objective To explore the upper signaling pathway in neutrophil elastase（NE）-induced mucinSAC（MUCSAC） production in human BEAS-2B airway epithelial cells. Methods The BEAS-2B airway epithelial cells were cultured and divided into 7 groups： negative control group, NE stimulation group, negative siRNA control group treated with or without NE, NADPH oxidase 1 （ Nox1 ） siRNA group treated with NE, c-src siRNA group treated with NE, and c-src specific inhibitor PP2 group treated NE. The relative content of reactive oxygen species （ROS） was assayed with a special kit. The levels of MUCSAC protein in culture medium, Nox1 protein, phosphoraiyted c-src （p-c-src） kinase and MUCSAC mRNA in culture cells were detected with enzyme-linked immunosorbent assay, Western blot, and RT-PCR, respectively. Results There was an obvious increase of ROS production （0. 76 ± 0. 09） in cells exposed to NE, with elevation of Nox1 protein （0. 88 0. 12） and MUCSAC protein production （0. 82 ± 0. 09） and mRNA expression （0. 77 ±0. 05 ）, all had significant differences when compared with normal control group, t = 6. 13-9.44, P 〈 0. 01. Noxl siRNA inhibited ROS activity （ 0. 19 ± 0. 06 ）, reduced MUC5AC protein （0. 31 ±0. 05） and mRNA level （0. 32 ±0. 06）, compared with single NE-stimulated group, P 〈0. 01. c- src siRNA and PP2 also showed the same effects, both of them decreased the NE-induced high activity of ROS, was 0. 29 ±0. 05, and 0. 41 ±0. 11, respectively, the MUC5AC protein in the c-sre siRNA group and PP2 group were 0. 38 ±0. 09 and 0.48 -＋0.08, MUCSAC mRNA level were 0. 41 ＋0. 04 and O. 46 -＋0. 07, the Noxl protein were 0. 39 -＋ 0. 08 and 0. 44 ＋ 0. 05, all P 〈 0. 05, compared with single NE-treated group. Conclusion C-sre/Noxl/ROS transduction cascade may be upper signal regulators in NE-mediated MUCSAC expression in BEAS-2B cells.
作者 李琪 周向东
机构地区 重庆医科大学附属第二医院呼吸内科
出处 《中华结核和呼吸杂志》 CAS CSCD 北大核心 2010年第1期46-50,共5页 Chinese Journal of Tuberculosis and Respiratory Diseases
基金 国家自然科学基金资助项目（30770951）
关键词 黏蛋白类 活性氧 自细胞弹性蛋白酶 NADPH氧化酶 src族激酶类 Mucins Reactive oxygen species Leukocyte elastase NADPH oxidase Src- family kinases
分类号 R562 [医药卫生—呼吸系统]
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参考文献9

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二级参考文献11

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共引文献7

同被引文献16

  • 1李琪,周向东.中性粒细胞弹力蛋白酶引起黏蛋白5AC高表达的信号转导机制[J].中华医学杂志,2007,87(5):348-352. 被引量：23
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中华结核和呼吸杂志
2010年 第1期

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