气道上皮细胞Src同源区2蛋白酪氨酸磷酸酶1基因敲除对慢性阻塞性肺疾病肺气肿表型的影响

Effect of SHP-1 knockout in airway epithelial cells on emphysema phenotype in chronic obstructive pulmonary disease in mice

目的构建并鉴定气道上皮细胞条件性Src同源区2蛋白酪氨酸磷酸酶1(SHP-1)基因敲除小鼠,观察慢性阻塞性肺疾病(慢阻肺)患者气道上皮SHP-1表达情况及SHP-1表达缺陷对肺气肿小鼠表型的影响。方法检测慢阻肺患者肺组织中气道上皮SHP-1的表达情况。采用CRISPR/Cas9技术构建SHP-1^(flox/flox)转基因小鼠,并将其与气道上皮细胞Clara蛋白10-环化重组酶与雌激素受体融合转基因小鼠(CC10-CreER^(+/+))进行交配,腹腔注射他莫昔芬后,获得气道上皮SHP-1基因敲除小鼠(SHP-1^(flox/flox)CC10-CreER^(+/-),SHP-1^(Δ/Δ))。提取小鼠鼠尾和肺组织DNA,经PCR扩增后判别小鼠的基因型;通过qRT-PCR、Western blot、免疫荧光等方法验证气道上皮细胞SHP-1基因的敲除效果。使用弹性蛋白酶构建肺气肿小鼠模型,评估各组小鼠肺气肿严重程度。结果慢阻肺患者气道上皮SHP-1表达显著下调。基因型鉴定证实SHP-1^(Δ/Δ)小鼠表达CC10-CreER及SHP-1-flox。他莫昔芬诱导后,我们从DNA、RNA及蛋白水平证明SHP-1^(Δ/Δ)小鼠气道上皮细胞中SHP-1蛋白表达缺失,表明气道上皮细胞特异性SHP-1基因敲除小鼠构建成功。肺气肿动物模型中,SHP-1^(Δ/Δ)小鼠与对照组相比,肺气肿表型更为严重,表现为肺组织肺泡结构紊乱,肺泡壁破裂融合形成肺大疱。结论本研究成功建立并鉴定气道上皮细胞SHP-1基因敲除小鼠模型,为深入阐明SHP-1在慢阻肺进程中的作用及其机制提供了新的实验工具。

Objective To construct and characterize conditional Src homology region 2 protein tyrosine phosphatase 1(SHP-1)knockout mice in airway epithelial cells and to observe the effect of defective SHP-1 expression in airway epithelial cells on the emphysema phenotype in chronic obstructive pulmonary disease(COPD).Methods To detect the expression of SHP-1 in the airway epithelium of COPD patients.CRISPR/Cas9 technology was used to construct SHP-1^(flox/flox) transgenic mice,which were mated with airway epithelial Clara protein 10-cyclase recombinase and estrogen receptor fusion transgenic mice(CC10-CreER^(+/+)),and after intraperitoneal injection of tamoxifen,airway epithelial SHP-1 knockout mice were obtained(SHP-1^(flox/flox)CC10-CreER^(+/-),SHP-1^(Δ/Δ)).Mouse tail and lung tissue DNA was extracted and PCR amplified to discriminate the genotype of the mice;the knockout effect of SHP-1 gene in airway epithelial cells was verified by qRT-PCR,Western blotting,and immunofluorescence.In addition,an emphysema mouse model was constructed using elastase to assess the severity of emphysema in each group of mice.Results Airway epithelial SHP-1 was significantly downregulated in COPD patients.Genotyping confirmed that SHP-1^(Δ/Δ)mice expressed CC10-CreER and SHP-1-flox.After tamoxifen induction,we demonstrated the absence of SHP-1 protein expression in airway epithelial cells of SHP-1^(Δ/Δ)mice at the DNA,RNA,and protein levels,indicating that airway epithelial cell-specific SHP-1 knockout mice had been successfully constructed.In the emphysema animal model,SHP-1^(Δ/Δ)mice had a more severe emphysema phenotype compared with the control group,which was manifested by disorganization of alveolar structure in lung tissue and rupture and fusion of alveolar walls to form pulmonary alveoli.Conclusions The present study successfully established and characterized the SHP-1 knockout mouse model of airway epithelial cells,which provides a new experimental tool for the in-depth elucidation of the role of SHP-1 in the emphysema process of COPD and its mechanism.