目的 构建气管组织来源的气道类器官（AO），探究脂多糖（LPS）刺激后对该AO细胞存活及炎症因子表达的影响。方法 游离小鼠气道组织，在体外经机械分离和胶原酶消化为单细胞，Matrigel重悬后在3D环境下培养，待其自发形成具有空腔样囊状结构，通过叉头框蛋白J1（FoxJ1）、Muc5AC、P63、CC10免疫荧光染色鉴定该囊状结构的细胞组成。待稳定传代至第5代后，加入0（对照组）、1、5、10、20 μg·mL^-1的LPS刺激，通过活（钙黄绿素）、死（碘化丙啶）染色检测细胞的存活情况，通过实时荧光定量PCR（qRT-PCR）检测炎症因子白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）的mRNA表达。结果 原代上皮细胞经3D培养后，自发形成囊状空腔样结构，并随着培养时间的延长，可见球形结构逐步增大，表面凸起较多新的细胞团。免疫荧光染色发现该AO包含纤毛细胞（FoxJ1⁺）、杯状细胞（Muc5AC⁺）、基底细胞（P63⁺）、棒状细胞（CC10⁺）。LPS刺激后，与对照组比较，活死染色显示10 μg·mL^-1 LPS诱导细胞死亡，并显著上调IL-1β、IL-6和TNF-α的mRNA表达（P＜0.05、0.01）。结论 成功构建小鼠气管组织来源AO炎症模型，可用于评估气道上皮细胞与炎症间的相互作用。

Objective To construct tracheal organoids derived from tracheal tissues and investigate the effects of lipopolysaccharide (LPS) stimulation on cell survival and the expression of inflammatory cytokines in these organoids. Methods Mouse tracheal tissues were isolated, mechanically dissociated, and digested with collagenase to obtain single cells. These cells were resuspended in Matrigel and cultured in a 3D environment, allowing them to spontaneously form cyst-like structures with lumens. The cellular composition of these structures was identified through FoxJ1, Muc5AC, P63, and CC10 immunofluorescence staining. After stable passage to the fifth generation, LPS of 0 (control group), 1, 5, 10, and 20 μg·mL^-1 were added. Cell survival was assessed by live (calcein)-dead (PI) staining, and the expression of related inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were detected by qRT-PCR to evaluate the impact of LPS on the function of the organoid cells. Results Primary epithelial cells spontaneously formed cyst-like structures with lumens after 3D culture. Over time, these spherical structures enlarged and displayed multiple new cell clusters on the surface. Immunofluorescence staining revealed that the organoids contained ciliated cells (FoxJ1⁺), goblet cells (Muc5AC⁺), club cells (P63⁺), and basal cells (CC10⁺). Following LPS stimulation, compared to control group, live-dead staining showed that high concentrations of LPS induced cell death and upregulated the expression of IL-1β, IL-6, and TNF-α significantly (P < 0.05 and 0.01). Conclusion A mouse tracheal organoid inflammatory model was successfully constructed that can be used to evaluate the interactions between airway epithelial cells and inflammation.

江西省技术创新引导类计划项目（S2023KJHZH0014）