实用医学杂志 ›› 2023, Vol. 39 ›› Issue (22): 2921-2927.doi: 10.3969/j.issn.1006-5725.2023.22.011

• 基础研究 • 上一篇    下一篇

慢性阻塞性肺疾病大鼠气道成纤维细胞过表达HSG基因对Wnt和MAPK信号通路的影响

杨义1,2,周洵2,张军2,李波2,王新星3,罗弦2,葛正行2()   

  1. 1.贵州中医药大学 (贵阳 550025 )
    2.贵州中医药大学第二附属医院呼吸内科
    3.贵州中医药大学第二附属医院科研教学部 (贵阳 550003 )
  • 收稿日期:2023-06-12 出版日期:2023-11-25 发布日期:2023-12-11
  • 通讯作者: 葛正行 E-mail:gezhengxing22@163.com
  • 基金资助:
    国家自然科学基金项目(81473533);贵州省重大应用基础研究(黔科合J重大字[2015]2002)

Effects of overexpression of HSG gene on Wnt signaling pathway and MAPK signaling pathway in COPD rat airway fibroblasts

Yi YANG1,2,Xun ZHOU2,Jun ZHANG2,Bo LI2,Xinxing WANG3,Xian LUO2,Zhengxing. GE2()   

  1. Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China;,
    Department of Respiratory Medicine,the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550003,China
  • Received:2023-06-12 Online:2023-11-25 Published:2023-12-11
  • Contact: Zhengxing. GE E-mail:gezhengxing22@163.com

摘要:

目的 研究在慢性阻塞性肺疾病(chronic obstructive pulmoriary disease, COPD)大鼠气道成纤维细胞中过表达HSG基因(MFN2)对Wnt信号通路的影响。 方法 采用蛋白酶诱导的方式构建大鼠COPD 模型,然后原代分离 COPD 模型大鼠气道成纤维细胞。构建HSG基因的表达载体,转染大鼠气道成纤维细胞,得到过表达HSG的大鼠气道成纤维细胞。实验一,采用Wnt信号途径激动剂佛波醇酯(TPA)处理细胞, WB、qPCR检测转染后HSG、Wnt5a的表达;实验二,采用MAPK 信号途径激动剂 EGF 因子处理细胞, WB、qPCR检测转染后HSG、ERK1/2的表达;而且每个实验都进行ELISA检测细胞培养液中TGF-β1、PDGF、MMP-9 的含量,流式检测细胞凋亡情况。 结果 COPD模型大鼠气道成纤维细胞分离成功。实验一二中, WB、qPCR检测显示过表达HSG能抑制基因Wnt5a和ERK1/2的表达,受相应激动剂处理后,这些基因表达上调而HSG表达受抑制;而ELISA检测结果显示,过表达HSG能降低TGF-β1、PDGF、MMP-9 的含量,受相应激动剂处理后TGF-β1、PDGF、MMP-9 的含量上升,而过表达HSG依然有抑制效果。流式检测细胞凋亡显示过表达HSG会明显上调细胞凋亡率,而相应的激动剂处理会降低过表达HSG促进细胞凋亡的效果。流式检测细胞周期显示过表达HSG对细胞周期的影响并不明显。 结论 在COPD 大鼠气道成纤维细胞中过表达HSG基因对Wnt和MAPK 等信号途径均有抑制效果,而且过表达HSG基因有着促进成纤维细胞凋亡,可以推测促进HSG表达具有抑制COPD大鼠气道纤维化的效果。

关键词: 慢性阻塞性肺疾病, Wnt信号通路, 成纤维细胞, HSG, MAPK信号通路

Abstract:

Objective To study the effect of overexpression of HSG gene (MFN2) on Wnt signaling pathway in airway fibroblasts of rats with chronic obstructive pulmonary disease (COPD). Methods The rat COPD model was induced by protease, and then the airway fibroblasts of COPD model rats were isolated in primary culture. The expression vector of HSG gene was constructed and transfected into rat airway fibroblasts to obtain rat airway fibroblasts overexpressing HSG. In Experiment 1, cells were treated with phorbol ester (TPA), a Wnt signaling pathway agonist, and the expressions of HSG and Wnt5a were detected by WB and qPCR; In Experiment 2, the cells were treated with EGF factor, an agonist of MAPK signal pathway, and the expressions of HSG and ERK1/2 were detected by WB and qPCR; Moreover, ELISA was used to detect TGF in cell culture medium in each experiment-β1. The contents of PDGF and MMP-9, CCK8, cell proliferation and apoptosis were detected by flow cytometry. Results Airway fibroblasts were successfully isolated from COPD model rats. In Experiment 1 and Experiment 2, WB and qPCR showed that overexpression of HSG could inhibit the expression of genes Wnt5a and ERK1/2. After treatment with corresponding agonists, the expression of these genes was up-regulated and the expression of HSG was inhibited; The results of ELISA showed that overexpression of HSG could reduce TGF-β1. The contents of PDGF and MMP-9, TGF after treatment with corresponding agonists-β1. The contents of PDGF and MMP-9 increased, but overexpression of HSG still had inhibitory effect. CCK8 detection showed that overexpression of HSG could inhibit cell proliferation, while flow cytometry showed that overexpression of HSG could significantly increase the apoptosis rate, and the corresponding agonist treatment would reduce the effect of overexpression of HSG on cell apoptosis. Flow cytometry showed that the effect of overexpression of HSG on cell cycle was not obvious. Conclusion Overexpression of HSG gene in airway fibroblasts of COPD rats can inhibit Wnt, MAPK and other signal pathways, and overexpression of HSG gene can promote fibroblast apoptosis and inhibit cell proliferation. It can be speculated that promoting HSG expression can inhibit airway fibrosis of COPD rats.

Key words: chronic obstructive pulmoriary disease, Wnt signaling pathway, fibroblast, HSG, MAPK signaling pathway

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