气道类器官研究哮喘中Lkb1调控上皮再生的机制  

Study of Lkb1 regulates epithelial regeneration in asthma using airway organoid

作　　者：徐桂颖 李玉[2,3] 李雪 刘怡萌[1] 陈怀永 XU Guiying;LI Yu;LI Xue;LIU Yimeng;CHEN Huaiyong(Haihe Clinical School,Tianjin Medical University,Tianjin 300350,China;Haihe Hospital Affiliated to Tianjin University;Tianjin Institute of Respiratory Diseases)

机构地区：[1]天津医科大学海河临床学院,300350 [2]天津大学海河医院 [3]天津市呼吸疾病研究所

出　　处：《天津医药》2024年第1期11-15,共5页Tianjin Medical Journal

基　　金：国家自然科学基金面上项目(82070001)。

摘　　要：目的 通过气道类器官培养研究哮喘中肝激酶B1(Lkb1)调控上皮再生的机制。方法 取Lkb1~(f/f)(对照组,10只)和Scgb1a1~(CreER);Lkb1~(f/f)小鼠(Lkb1敲除组,9只),采用雾化吸入鸡卵清蛋白(OVA)的方法建立过敏性哮喘模型,收集支气管肺泡灌洗液(BALF)和肺组织,统计BALF中炎性细胞数量,肺组织切片免疫荧光染色比较钙激活氯离子通道蛋白3(CLCA3)阳性细胞数量。通过流式细胞术分选出Club细胞进行类器官培养,统计类器官的平均直径和类器官形成率,回收细胞通过实时荧光定量PCR(RT-PCR)技术检测高脚杯细胞标志物CLCA3、纤毛细胞标志物叉头框蛋白J1(FOXJ1)和Club细胞中腺苷酸活化蛋白激酶(AMPK)的表达水平。结果 与对照组相比,Lkb1敲除组BALF中巨噬细胞、嗜酸性粒细胞、中性粒细胞和淋巴细胞数量变化差异无统计学意义;Lkb1敲除后CLCA3阳性细胞数量减少;类器官培养结果显示敲除Lkb1后Club细胞来源的类器官平均直径减小,类器官形成率降低,纤毛细胞分化标志物FOXJ1 m RNA表达水平降低,缺失Lkb1后Club细胞表达AMPKα水平降低,且Club细胞增殖受到抑制,激活Lkb1的下游信号通路AMPK可以减弱Lkb1缺失对Club细胞再生功能的影响。结论 Lkb1通过AMPK通路促进气道祖细胞增殖。Objective To explore the mechanism of Lkb1 regulated epithelial regeneration in asthma by airway organoid culture.Methods Lkb1f/f(the control group,n=10)and Scgb1a1CreER;Lkb1f/f mice(the Lkb1 knockout group,n=9)were taken to establish allergic asthma models by aerosol inhalation of ovalbumin(OVA).Bronchial lavage fluid(BALF)and lung tissue were collected.The number of inflammatory cells in BALF were counted.The amount of CLCA3 positive cells was compared by immunofluorescence staining of lung tissue sections.Club cells were selected by flow cytometry for organoid culture.The average diameter of organoids and organoid formation rate were calculated.Expression levels of goblet cell marker CLCA3,cilia cell markers FOXJ1 and AMPK in Club cells were detected by RT-PCR.Results There were no significant differences in the number of macrophages,eosinophils,neutrophils and lymphocytes in BALF between the control group and the Lkb1 knockout group.The number of CLCA3 positive cells were decreased after Lkb1 knockout.Results of organoid culture showed that the average diameter of organoids derived from Club cells and organoid formation rate were decreased after the absence of Lkb1.The expression of FOXJ1 was reduced.After Lkb1 deletion,the expression of AMPKαin Club cells were decreased and the proliferation of Club cells was inhibited.Activation of AMPK,the downstream signaling pathway of Lkb1,could attenuate the effect of Lkb1 deficiency on the regeneration of Club cells.Conclusion Lkb1 promotes the proliferation of airway progenitor cells by AMPK pathway.

关 键 词：肝激酶B1 气道祖细胞 腺苷酸活化蛋白激酶 类器官培养 哮喘 细胞增殖 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]