NO介导的代谢紊乱影响小鼠气道祖细胞增殖的机制研究

doi:10.11958/20222109

天津医药 ›› 2023, Vol. 51 ›› Issue (5): 477-481.doi: 10.11958/20222109

NO介导的代谢紊乱影响小鼠气道祖细胞增殖的机制研究

郝德(), 李宽, 王建海, 岳青, 陈怀永^△()

天津大学海河医院，天津市肺脏再生医学重点实验室，天津市呼吸疾病研究所（邮编300350）

收稿日期:2022-12-28 修回日期:2023-02-10 出版日期:2023-05-15 发布日期:2023-05-05
通讯作者: △E-mail:huaiyong.chen@foxmail.com
作者简介:郝德（1995），男，硕士研究生，主要从事肺损伤与修复机制方面研究。E-mail：2020235073@tju.edu.cn
基金资助:
国家自然科学基金面上项目(82070001)

Effects of nitric oxide-mediated metabolic disruption on the proliferation of mouse airway progenitor cells

HAO De(), LI Kuan, WANG Jianhai, YUE Qing, CHEN Huaiyong^△()

Tianjin Key Laboratory of Lung Regenerative Medicine, Haihe Hospital, Tianjin University, Tianjin 300350, China

Received:2022-12-28 Revised:2023-02-10 Published:2023-05-15 Online:2023-05-05
Contact: △E-mail:huaiyong.chen@foxmail.com

郝德, 李宽, 王建海, 岳青, 陈怀永. NO介导的代谢紊乱影响小鼠气道祖细胞增殖的机制研究[J]. 天津医药, 2023, 51(5): 477-481.

HAO De, LI Kuan, WANG Jianhai, YUE Qing, CHEN Huaiyong. Effects of nitric oxide-mediated metabolic disruption on the proliferation of mouse airway progenitor cells[J]. Tianjin Medical Journal, 2023, 51(5): 477-481.

摘要/Abstract

摘要：

目的研究一氧化氮（NO）对小鼠气道club祖细胞增殖功能的调控及可能代谢机制。方法使用流式分选技术分选小鼠气道原代club祖细胞，经25 μmol/L的NO供体二乙胺壬酸酯（DEA NONOate）处理后进行转录组测序；分选小鼠气道原代club祖细胞，使用肺脏类器官技术体外培养原代club祖细胞，分别加入正常培养基，40 nmol/L辛伐他汀（Hmgcr抑制剂），0.2 μmol/L 6-重氮-5-氧代-L-正亮氨酸（DON，Gmps抑制剂），1 mmol/L氨基羟乙酸酯（AOA，Gpt2抑制剂）培养基，在培养第8天时使用倒置显微镜拍照，观察原代club祖细胞衍生类器官生长情况，并统计类器官直径和形成效率。结果 DEA NONOate处理原代club祖细胞转录组表达显著变化（上调倍数超过1.2倍的基因2 894个，下调16.7%以上的基因3 270个），其中代谢相关基因Hmgcr、Gmps和Gpt2的表达量显著下降（P<0.01）。与对照组相比，辛伐他汀组原代club祖细胞形成的类器官直径减小（P<0.01），类器官形成效率没有变化；DON组类器官直径减小（P<0.01），类器官形成效率降低（P<0.01）；AOA组类器官直径减小（P<0.01），类器官形成效率降低（P<0.01）。结论 NO抑制小鼠气道原代club祖细胞增殖功能可能与其负调控Hmgcr、Gmps和Gpt2代谢相关通路有关。

关键词: 一氧化氮, 细胞增殖, 类器官, 气道祖细胞, 细胞代谢

Abstract:

Objective To evaluate the effect of nitric oxide (NO) on the proliferative function of airway club progenitor cells and underlying metabolic mechanisms. Methods The mouse airway progenitor club cells were sorted using flow sorting technique, then treated with 25 μmol/L DEA NONOate (diethylaminononanoate) for transcriptomic sequencing analysis. Mouse club cells were cultured using organoid culture technique. The cells were cultured in normal medium, 40 nmol/L simvastain (Hmgcr inhibitor), 0.2 μmol/L DON (Gmps inhibitor) or 1 mmol/L AOA (Gpt2 inhibitor) respectively. Microscopic photographs were taken with an inverted microscope on the day 8 of culture to observe the growth of club cell-derived organoids and to measure the organoid diameter and formation efficiency. Results DEA NONOate treatment of club cells resulted in significant changes in transcriptome expression (2 894 up-regulated genes and 3 270 down-regulated genes, fold ≥ 1.2), with significant decrease in expression levels of metabolism-related genes Hmgcr, Gmps and Gpt2 (P<0.01). Compared with the control group, the organoid diameter was significantly reduced (P<0.01), but the organoid forming efficiency was unchanged in the simvastain-treated group. Both organoid diameter and forming efficiency were significantly reduced in the DON-treated group (P<0.01) and the AOA-treated group (P<0.01). Conclusion NO inhibits the proliferative function of mouse airway club progenitor cells probably through its negative regulation of three metabolism-related pathways, Hmgcr, Gmps and Gpt2.

Key words: nitric oxide, cell proliferation, organoids, airway epithelial progenitor cells, cell metabolism

中图分类号:

R329.2+8，R562.2

图/表 6

图1 小鼠气道原代club祖细胞流式分选策略与类器官培养模型

Fig.1 Flow sorting strategy of mouse airway club cells and organoid culture model

图2 NO刺激后原代club祖细胞差异变化基因火山图

Fig.2 The differential change genes of club cells after NO stimulation

图3 NO对小鼠原代club祖细胞相关代谢基因表达的影响

Fig.3 The effect of NO on the expression of metabolic genes in mouse club cells

图4 辛伐他汀对小鼠原代club祖细胞增殖功能的影响（×40）

Fig.4 The effect of simvastain on the size of mouse club cell-derived organoids (×40)

图5 DON对小鼠原代club祖细胞增殖功能的影响（×40）

Fig.5 The effect of DON on the size of mouse club cell-derived organoids (×40)

图6 AOA对小鼠原代club祖细胞增殖功能的影响（×40）

Fig.6 The effect of AOA on the size of mouse club cell-derived organoids (×40)

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NO介导的代谢紊乱影响小鼠气道祖细胞增殖的机制研究

Effects of nitric oxide-mediated metabolic disruption on the proliferation of mouse airway progenitor cells

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