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

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25µmol/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),0.2µmol/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2894个,下调16.7%以上的基因3270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。

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 25µmol/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),0.2µmol/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 cellderived organoids and to measure the organoid diameter and formation efficiency.Results DEA NONOate treatment of club cells resulted in significant changes in transcriptome expression(2894 up-regulated genes and 3270 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 metabolismrelated pathways,Hmgcr,Gmps and Gpt2.