目的 構(gòu)建氣管組織來(lái)源的氣道類(lèi)器官（AO），探究脂多糖（LPS）刺激后對(duì)該AO細(xì)胞存活及炎癥因子表達(dá)的影響。方法 游離小鼠氣道組織，在體外經(jīng)機(jī)械分離和膠原酶消化為單細(xì)胞，Matrigel重懸后在3D環(huán)境下培養(yǎng)，待其自發(fā)形成具有空腔樣囊狀結(jié)構(gòu)，通過(guò)叉頭框蛋白J1（FoxJ1）、Muc5AC、P63、CC10免疫熒光染色鑒定該囊狀結(jié)構(gòu)的細(xì)胞組成。待穩(wěn)定傳代至第5代后，加入0（對(duì)照組）、1、5、10、20 μg·mL^-1的LPS刺激，通過(guò)活（鈣黃綠素）、死（碘化丙啶）染色檢測(cè)細(xì)胞的存活情況，通過(guò)實(shí)時(shí)熒光定量PCR（qRT-PCR）檢測(cè)炎癥因子白細(xì)胞介素（IL）-1β、IL-6、腫瘤壞死因子-α（TNF-α）的mRNA表達(dá)。結(jié)果 原代上皮細(xì)胞經(jīng)3D培養(yǎng)后，自發(fā)形成囊狀空腔樣結(jié)構(gòu)，并隨著培養(yǎng)時(shí)間的延長(zhǎng)，可見(jiàn)球形結(jié)構(gòu)逐步增大，表面凸起較多新的細(xì)胞團(tuán)。免疫熒光染色發(fā)現(xiàn)該AO包含纖毛細(xì)胞（FoxJ1⁺）、杯狀細(xì)胞（Muc5AC⁺）、基底細(xì)胞（P63⁺）、棒狀細(xì)胞（CC10⁺）。LPS刺激后，與對(duì)照組比較，活死染色顯示10 μg·mL^-1 LPS誘導(dǎo)細(xì)胞死亡，并顯著上調(diào)IL-1β、IL-6和TNF-α的mRNA表達(dá)（P＜0.05、0.01）。結(jié)論 成功構(gòu)建小鼠氣管組織來(lái)源AO炎癥模型，可用于評(píng)估氣道上皮細(xì)胞與炎癥間的相互作用。

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.

江西省技術(shù)創(chuàng)新引導(dǎo)類(lèi)計(jì)劃項(xiàng)目（S2023KJHZH0014）