Xu S L, Chen Z, Yang J, Fan Z Q, Liu T, Zhang X T, Zeng B Q, Xing X Q
Department of Respiratory and Critical Care Medicine, The Affiliated Hospital of Yunnan University, The Second People's Hospital of Yunnan Province, Kunming 650021, Yunnan, China.
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2024 Apr 12;47(4):339-345. doi: 10.3760/cma.j.cn112147-20231114-00309.
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). To detect the expression of SHP-1 in the airway epithelium of COPD patients. CRISPR/Cas9 technology was used to construct SHP-1flox/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-1CC10-CreER, SHP-1). 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. Airway epithelial SHP-1 was significantly downregulated in COPD patients. Genotyping confirmed that SHP-1 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 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 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. 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.
构建并鉴定气道上皮细胞中条件性Src同源区2蛋白酪氨酸磷酸酶1（SHP-1）基因敲除小鼠，观察气道上皮细胞中SHP-1表达缺陷对慢性阻塞性肺疾病（COPD）肺气肿表型的影响。检测COPD患者气道上皮中SHP-1的表达。利用CRISPR/Cas9技术构建SHP-1flox/flox转基因小鼠，将其与气道上皮克拉拉细胞蛋白10-环化酶重组酶和雌激素受体融合转基因小鼠（CC10-CreER）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-1CC10-CreER，SHP-1）。提取小鼠尾和肺组织DNA并进行PCR扩增以鉴别小鼠基因型；通过qRT-PCR、蛋白质免疫印迹和免疫荧光验证气道上皮细胞中SHP-1基因的敲除效果。此外，用弹性蛋白酶构建肺气肿小鼠模型，评估每组小鼠肺气肿的严重程度。COPD患者气道上皮SHP-1明显下调。基因分型证实SHP-1小鼠表达CC10-CreER和SHP-
