沉默Rho GDIα 抑制矽肺上皮间质转化及其作用机制
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(1.华北理工大学基础医学院,河北唐山 063000; 2.华北理工大学医学实验研究中心,河北唐山 063000;3.华北理工大学中医学院,河北唐山 063000)

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R-33

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Suppressive effect of Rho GDIα knockdown on epithelial-mesenchymal transition in silicosis and its mechanism
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(1. Basic Medical College, North China University of Science and Technology, Tangshan 063000, China.2. Medical Research Center, North China University of Science and Technology, Tangshan 063000.3. College of Traditional Chinese Medicine, North China University of Science and Technology, Tangshan 063000)

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    摘要:

    目的 探讨沉默Rho GDP 解离抑制因子α(Rho GDP dissociation inhibitor α, Rho GDIα)对矽肺上皮间质转化的作用及机制。方法 转化生长因子-β1(transforming growth factor-β1, TGF-β1)诱导人肺泡Ⅱ型上皮细胞株A549,分为空载体慢病毒组(LEV)、沉默Rho GDIα 慢病毒感染组(Lv-Rho GDIα-inhibition)、TGF-β1 诱导LEV组(LEV+TGF-β1)、TGF-β1 诱导Lv-Rho GDIα-inhibition 组(Lv-Rho GDIα-inhibition+TGF-β1)。采用Western blot法、免疫细胞化学染色检测Rho GDIα、RhoA、Rho 激酶(Rho kinase, ROCK)、E-钙黏蛋白(E-cadherin, E-cad)、α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)和I 型胶原(collagen-I)蛋白表达,采用Western blot 法及CCK-8 检测细胞增殖能力。结果 Western blot 法及免疫细胞化学染色结果显示,与LEV 对照组相比,Lv-Rho GDIα-inhibition组Rho GDIα、RhoA、ROCK 蛋白表达下降,E-cad 蛋白表达上调,α-SMA 和collagen-I 蛋白表达下降; LEV+TGF-β1组Rho GDIα、RhoA、ROCK 蛋白表达上升,E-cad 蛋白表达下调,α-SMA 和collagen-I 蛋白表达上升。与LEV+TGF-β1 组相比,Lv-Rho GDIα-inhibition+TGF-β1 组Rho GDIα、RhoA、ROCK 蛋白表达下降,E-cad 蛋白表达上调,α-SMA和collagen-I 蛋白表达下降。CCK-8 及cyclin D1 蛋白检测结果显示,与LEV 对照组相比,Lv-Rho GDIα-inhibition 组细胞增殖受到抑制,LEV+TGF-β1 组细胞增殖增强,与LEV+TGF-β1 组相比,Lv-Rho GDIα-inhibition+TGF-β1 组细胞增殖受到抑制。结论 沉默Rho GDIα 可通过调控RhoA/ ROCK 信号通路抑制上皮间质转化发挥抗矽肺纤维化作用。

    Abstract:

    Objective To explore the effect and mechanism of Rho GDP dissociation inhibitor α (Rho GDIα) knockdown on epithelial-mesenchymal transition (EMT) in silicosis. Methods The alveolar type Ⅱ epithelial cell line A549 was induced by transforming growth factor-β1(TGF-β1) for EMT.The cells were divided into four groups: lentivirus empty vector (LEV) group, RhoGDIα depletion lentiviral vector (Lv-Rho GDIα-inhibition)group, LEV-transfected cells induced by TGF-β1 (LEV+TGF-β1) group, and Lv-Rho GDIα-inhibition transfected cells induced by TGF-β1 (Lv-Rho GDIα-inhibition+TGF-β1) group. Western blotting and immunocytochemical staining were used to examine the protein expression of Rho GDIα, RhoA, Rho kinase(ROCK), E-cadherin (E-cad), α-smooth muscle actin(α-SMA) and collagen-I. Results Compared with the LEV control group, Rho GDIα, RhoA, ROCK, α-SMA and collagen-I showed upregulated expression, while E-cad showed downregulated expression in the Lv-Rho GDIα-inhibition group. Compared with the LEV + TGF-β1 group, Rho GDIα, RhoA, ROCK, α-SMA and collagen-I were all upregulated, and E-cad was downregulated in the Lv-Rho GDIα-inhibition+TGF-β1 group. Western blotting and Cell Counting Kit-8 result showed that silencing of Rho GDIα inhibited cell proliferation. Conclusions Rho GDIα knockdown may lead to the inhibition of epithelial-mesenchymal transformation by regulating the RhoA/ ROCK signaling pathway, and plays an anti-silicosis effect.

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耿菲,陈莹莹,姚婧昕,李世峰,高学敏,徐丁洁,魏中秋,杨方,徐洪.沉默Rho GDIα 抑制矽肺上皮间质转化及其作用机制[J].中国比较医学杂志,2019,29(12):10~15.

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  • 收稿日期:2019-07-08
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  • 在线发布日期: 2020-01-13
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