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不同分化阶段人气管类器官呼吸道合胞病毒感染后差异性特征

Differential Characteristics of Human Airway Organoids at Different Stages of Differentiation After Respiratory Syncytial Virus Infection

    摘要
  • 摘要:
    目的 探讨不同分化阶段人气管类器官呼吸道合胞病毒（respiratory syncytial virus, RSV）感染后的病变程度和免疫应答差异性特征。
    方法 通过构建人胚胎肺类器官和气管类器官模型模拟未成熟和成熟的气道上皮，用免疫荧光染色、电镜和实时荧光定量聚合酶链式反应（real-time fluorescence quantitative PCR, Q-PCR）技术验证上述类器官模型构建成功；用RSV感染不同成熟度的人肺类器官，分别在感染6 h和48 h收集肺类器官样本，通过免疫荧光染色、微滴式数字化聚合酶链式反应（droplet digital PCR, DDPCR）和Q-PCR技术探究RSV感染不同成熟度肺类器官的免疫特点。
    结果 成功构建了性别决定相关基因簇2（sex determining region Y-box transcription factor 2, SOX2）和性别决定相关基因簇9（sex determining region Y-box transcription factor 9, SOX9）双阳性的胚胎肺类器官以及包含基底细胞、纤毛细胞、club细胞和杯状细胞的成熟气管类器官模型。同时，利用RSV建立了类器官RSV感染模型。DDPCR结果显示，在RSV感染初期气管类器官中的病毒载量高于胚胎肺类器官（P<0.001），但RSV感染48 h气管类器官中RSV病毒载量低于胚胎肺类器官（P<0.05）。Q-PCR结果显示，气管类器官中RSV感染受体基因表达量高于胚胎肺类器官，如表皮生长因子受体（epidermal growth factor receptor, EGFR）、胰岛素样生长因子1受体（insulin-like growth factor 1 receptor, IGF1R）、核仁素（nucleolin, NCL），差异均有统计学意义（P<0.001）。同时在RSV感染48 h后气管类器官中免疫因子表达量较胚胎肺类器官升高，如白细胞介素6（interleukin 6, IL-6）、白细胞介素8（interleukin 8, CXCL8）、干扰素α（interferon α, IFN-α）、粒细胞集落刺激因子（granulocyte colony-stimulating factor, G-CSF）、粒细胞-巨噬细胞集落刺激因子（granulocyte macrophage colony-stimulating factor, GM-CSF）、肿瘤坏死因子α（tumor necrosis factor α, TNF-α），差异均有统计学意义（P<0.05)。
    结论 气道发育成熟会增加病毒感染受体蛋白表达，成熟气道上皮细胞比未成熟状态具有更强的免疫应答，抑制RSV病毒复制。

     

    Abstract:
    Objective To investigate the differences in pathological changes and immune responses of human airway organoids at different stages of differentiation following respiratory syncytial virus (RSV) infection.
    Methods Models of human fetal lung organoids (FLO) and induced airway organoids (iAO) were established to simulate immature and mature airway epithelium. Immunofluorescence staining, electron microscopy, and quantitative polymerase chain reaction (Q-PCR) were used to confirm the successful construction of the lung organoid models. Human lung organoids were infected with RSV, and samples were collected at 6 and 48 hours post-infection. The immune characteristics of immature and mature RSV-infected organoids were assessed using immunofluorescence staining, droplet digital PCR (DDPCR), and Q-PCR.
    Results We successfully generated FLO expressing both the progenitor markers sex determining region Y-box transcription factor 2 (SOX2) and sex determining region Y-box transcription factor 9 (SOX9), as well as iAO containing basal cells, ciliated cells, club cells, and goblet cells. In addition, organoid models of RSV infection were established. DDPCR results showed that, at the initial stage of RSV infection, the viral load in iAO was significantly higher than that in FLO (P < 0.001). However, at 48 hours post-infection, the viral load in iAO was lower than that in FLO (P < 0.05). Q-PCR results indicated that the expression of RSV infection receptor genes, including epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), and nucleolin (NCL), was significantly higher in iAO compared to that in FLO (P < 0.001). RSV infection led to an increase in the expression levels of immune factors, including interleukin 6 (IL-6), interleukin 8 (CXCL8), interferon α (IFN-α), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor α (TNF-α), in iAO compared to those in FLO, and the differences were statistically significant (P < 0.05).
    Conclusion The expression of RSV infection receptor proteins increases with airway maturation, and mature airway epithelial cells exhibit a stronger immune response than immature ones do, effectively inhibiting RSV replication.

     

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