Study of Lkb1 regulates epithelial regeneration in asthma using airway organoid

doi:10.11958/20231506

Abstract

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

CLC Number:

R329.2+8

Figures/Tables 5

References 28

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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