气道类器官研究哮喘中Lkb1调控上皮再生的机制

doi:10.11958/20231506

摘要/Abstract

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 mRNA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。

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

Abstract:

Objective To explore the mechanism of Lkb1 regulated epithelial regeneration in asthma by airway organoid culture. Methods Lkb1^f/f (the control group, n=10) and Scgb1a1^CreER; Lkb1^f/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 AMPKα in 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.

Key words: liver kinase B1, airway progenitor cells, AMP-activated protein kinase, organoid culture, asthma, cell proliferation

中图分类号:

R329.2+8

图/表 5

参考文献 28

[1]	BARNES P J. Immunology of asthma and chronic obstructive pulmonary disease[J]. Nat Rev Immunol, 2008, 8(3):183-192. doi:10.1038/nri2254.
[2]	BAYARRI M A, MILARA J, ESTORNUT C, et al. Nitric oxide system and bronchial epithelium: more than a barrier[J]. Front Physiol, 2021, 12:687381. doi:10.3389/fphys.2021.687381.
[3]	JEFFERY P K, WARDLAW A J, NELSON F C, et al. Bronchial biopsies in asthma. An ultrastructural,quantitative study and correlation with hyperreactivity[J]. Am Rev Respir Dis, 1989, 140(6):1745-1753. doi:10.1164/ajrccm/140.6.1745.
[4]	ZHANG J T, ZHANG D, PAN Y, et al. The TL1A-DR3 axis in asthma: membrane-bound and secreted TL1A co-determined the development of airway remodeling[J]. Allergy Asthma Immunol Res, 2022, 14(2):233-253. doi:10.4168/aair.2022.14.2.233.
[5]	SUMBLY V, LANDRY I. Unraveling the role of STK11/LKB1 in non-small cell cung cancer[J]. Cureus, 2022, 14(1):e21078. doi:10.7759/cureus.21078.
[6]	CHEN G, KORFHAGEN T R, XU Y, et al. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production[J]. J Clin Invest, 2009, 119(10):2914-2924. doi:10.1172/JCI39731.
[7]	READER J R, TEPPER J S, SCHELEGLE E S, et al. Pathogenesis of mucous cell metaplasia in a murine asthma model[J]. Am J Pathol, 2003, 162(6):2069-2078. doi:10.1016/S0002-9440(10)64338-6.
[8]	VIAU A, BIENAIME F, LUKAS K, et al. Cilia-localized LKB1 regulates chemokine signaling,macrophage recruitment,and tissue homeostasis in the kidney[J]. EMBO J, 2018, 37(15):e98615. doi:10.15252/embj.201798615.
[9]	SAITO Y, NAKADA D. The role of the Lkb1/AMPK pathway in hematopoietic stem cells and Leukemia[J]. Crit Rev Oncog, 2014, 19(5):383-397. doi:10.1615/critrevoncog.2014011765.
[10]	NAKADA D, SAUNDERS T L, MORRISON S J. Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells[J]. Nature, 2010, 468(7324):653-658. doi:10.1038/nature0957.
[11]	GAN B, HU J, JIANG S, et al. Lkb1 regulates quiescence and metabolic homeostasis of haematopoietic stem cells[J]. Nature, 2010, 468(7324):701-704. doi:10.1038/nature09595.
[12]	MAILLET V, BOUSSETTA N, LECLERC J, et al. LKB1 as a gatekeeper of hepatocyte proliferation and genomic integrity during liver regeneration[J]. Cell Rep, 2018, 22(8):1994-2005. doi:10.1016/j.celrep.2018.01.086.
[13]	SHAN T, ZHANG P, LIANG X, et al. Lkb1 is indispensable for skeletal muscle development,regeneration,and satellite cell homeostasis[J]. Stem Cells, 2014, 32(11):2893-2907. doi:10.1002/stem.1788.
[14]	LI Y, ZHANG Q, LI L, et al. LKB1 deficiency upregulates RELM-alpha to drive airway goblet cell metaplasia[J]. Cell Mol Life Sci, 2021, 79(1):42. doi:10.1007/s00018-021-04044-w.
[15]	LI X, WU J, SUN X, et al. Autophagy reprograms alveolar progenitor cell metabolism in response to lung injury[J]. Stem Cell Reports, 2020, 14(3):420-432. doi:10.1016/j.stemcr.2020.01.008.
[16]	BONANNO L, ZULATO E, PAVAN A, et al. LKB1 and tumor metabolism:the interplay of immune and angiogenic microenvironment in lung cancer[J]. Int J Mol Sci, 2019, 20(8):1874. doi:10.3390/ijms20081874.
[17]	SANCHEZ-CESPEDES M. The role of LKB1 in lung cancer[J]. Fam Cancer, 2011, 10(3):447-453. doi:10.1007/s10689-011-9443-0.
[18]	FREY A, LUNDING L P, EHLERS J C, et al. More than just a barrier:the immune functions of the airway epithelium in asthma pathogenesis[J]. Front Immunol, 2020, 11:761. doi:10.3389/fimmu.2020.00761.
[19]	CANAS J A, SASTRE B, RODRIGO-MUNOZ J M, et al. Eosinophil-derived exosomes contribute to asthma remodelling by activating structural lung cells[J]. Clin Exp Allergy, 2018, 48(9):1173-1185. doi:10.1111/cea.13122.
[20]	DUTTA D, HEO I, CLEVERS H. Disease modeling in stem cell-derived 3D organoid systems[J]. Trends Mol Med, 2017, 23(5):393-410. doi:10.1016/j.molmed.2017.02.007.
[21]	ARTEGIANI B, CLEVERS H. Use and application of 3D-organoid technology[J]. Hum Mol Genet, 2018, 27(R2):R99-R107. doi:10.1093/hmg/ddy187.
[22]	CHEN J, NA F. Organoid technology and applications in lung diseases:models,mechanism research and therapy opportunities[J]. Front Bioeng Biotechnol, 2022, 10:1066869. doi:10.3389/fbioe.2022.1066869.
[23]	WANG J, LI X, CHEN H. Organoid models in lung regeneration and cancer[J]. Cancer Lett, 2020, 475:129-135. doi:10.1016/j.canlet.2020.01.030.
[24]	CHU Q, YAO C, QI X, et al. STK11 is required for the normal program of ciliated cell differentiation in airways[J]. Cell Discovery, 2019, 5:36. doi:10.1038/s41421-019-0104-z.
[25]	GAO Y, YAN Y, TRIPATHI S, et al. LKB1 represses ATOH1 via PDK4 and energy metabolism and regulates intestinal stem cell fate[J]. Gastroenterology, 2020, 158(5):1389-1401.e10. doi:10.1053/j.gastro.2019.12.033.
[26]	HAN Y, FENG H, SUN J, et al. Lkb1 deletion in periosteal mesenchymal progenitors induces osteogenic tumors through mTORC1 activation[J]. J Clin Invest, 2019, 129(5):1895-1909. doi:10.1172/JCI124590.
[27]	KARUMAN P, GOZANI O, ODZE R D, et al. The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death[J]. Mol Cell, 2001, 7(6):1307-1319. doi:10.1016/s1097-2765(01)00258-1.
[28]	MIHAYLOVA M M, SHAW R J. The AMPK signalling pathway coordinates cell growth,autophagy and metabolism[J]. Nat Cell Biol, 2011, 13(9):1016-1023. doi:10.1038/ncb2329.

基因名称	引物序列（5′→3′）	产物大小/ bp
β-actin	上游：GGCCAACCGTGAAAAGATGA 下游：CAGCCTGGATGGCTACGTACA	77
CLCA3	上游：GGCATCGTCATCGCCATAG 下游：CACCATGTCCTTTATGTGTTGAATG	72
FOXJ1	上游：AGAGAGTGAGGGCAAGAGAC 下游：GCGGGCTTAGAGACCATTTC	94
AMPKα	上游：GATCCTTTCCGGTGTGGATTAT 下游：GAAAGACCAAAGTCGGCTATCT	117
AMPKβ	上游：GAGATCAAGGCTCCAGAGAAAG 下游：GTTGAAGGACCCAGACAAGTAG	144
AMPKγ	上游：AGCCTGTTTGAAGCTGTCTATG 下游：CCGCTTGTGTGTGAGTATGTAG	105

基因名称	引物序列（5′→3′）	产物大小/ bp
β-actin	上游：GGCCAACCGTGAAAAGATGA 下游：CAGCCTGGATGGCTACGTACA	77
CLCA3	上游：GGCATCGTCATCGCCATAG 下游：CACCATGTCCTTTATGTGTTGAATG	72
FOXJ1	上游：AGAGAGTGAGGGCAAGAGAC 下游：GCGGGCTTAGAGACCATTTC	94
AMPKα	上游：GATCCTTTCCGGTGTGGATTAT 下游：GAAAGACCAAAGTCGGCTATCT	117
AMPKβ	上游：GAGATCAAGGCTCCAGAGAAAG 下游：GTTGAAGGACCCAGACAAGTAG	144
AMPKγ	上游：AGCCTGTTTGAAGCTGTCTATG 下游：CCGCTTGTGTGTGAGTATGTAG	105

组别	n	总细胞	巨噬细胞	嗜酸性粒细胞	中性粒细胞	淋巴细胞
对照组	10	10.500（6.300，29.025）	3.064（0.721，6.827）	5.804（0.386，17.072）	1.936（0.705，2.605）	1.342（0.631，1.856）
Lkb1敲除组	9	5.100（3.250，14.500）	1.810（0.833，3.685）	0.277（0.099，4.980）	1.401（0.253，2.278）	1.109（0.762，1.978）
Z		1.715	0.408	1.551	1.306	0.327

组别	n	总细胞	巨噬细胞	嗜酸性粒细胞	中性粒细胞	淋巴细胞
对照组	10	10.500（6.300，29.025）	3.064（0.721，6.827）	5.804（0.386，17.072）	1.936（0.705，2.605）	1.342（0.631，1.856）
Lkb1敲除组	9	5.100（3.250，14.500）	1.810（0.833，3.685）	0.277（0.099，4.980）	1.401（0.253，2.278）	1.109（0.762，1.978）
Z		1.715	0.408	1.551	1.306	0.327