Ⅱ型肺泡上皮细胞特异性敲除α-ENaC基因对成年小鼠肺水清除作用的研究

Pulmonary edema is a common clinical disease, for the treatment of which it is critical to remove the excessive fluid in the alveoli. In the early stage of pulmonary edema, the water reabsorption effect of type II alveolar epithelial cells plays an important role on the lung injury repair. The ENaC channel on the alveolar epithelial cells is responsible for the reabsorption of water and electrolytes, which is crucial for maintain alveolar fluid balance. α-ENaC is reported as a pore-forming subunit of ENaC. The general knock-out of mouse α-ENaC gene results in failure to clear fetal lung liquid at birth and in early neonatal. However, the functional study of α-ENaC gene in adult mouse is still limited. Thus in this project, we are firstly planning to establish an adult mouse model with ATⅡcell specific knock-out of α-ENaC gene using CRISPR/Cas9 and Cre/loxP systems. Then, the influence on alveolar fluid clearance, lung morphology wet/dry ratio and amiloride-sensitive sodium current will be further investigated within this mouse model, which will confirm the role of α-ENaC gene on alveolar fluid clearance in adult mouse and provide theoretical reference for clinical treatment of pulmonary edema.

肺水肿是临床上常见疾病，清除肺泡内过多液体是其治疗的关键。在肺水肿早期，II型肺泡上皮细胞(alveolar epithelial typeⅡcells，ATⅡ)对水的重吸收在肺损伤修复中具有重要作用。肺泡上皮细胞的ENaC通道负责水和电解质的重吸收，是保持肺泡液体平衡的重要离子通道。α-ENaC是ENaC的功能亚基，研究证实:全身性敲除α-ENaC基因后，新生小鼠出生后因不能有效清除肺内羊水而死亡，但对于成年小鼠的α-ENaC在肺泡中的作用研究较少。因此本课题拟通过CRISPR/Cas9和Cre/loxP技术建立ATⅡ细胞特异性α-ENaC基因敲除成年小鼠模型，使用该模型研究α-ENaC对成年小鼠肺水清除率、肺组织形态和干湿重比以及ATⅡ细胞的阿米洛利敏感性钠电流的影响，以确定α-ENaC在成年小鼠肺水清除中的作用，为临床治疗肺水肿提供理论参考。

肺水肿是临床上常见疾病，清除肺泡内过多液体是其治疗的关键。在肺水肿早期，II型肺泡上皮细胞(alveolar epithelial typeⅡcells，ATⅡ)对水的重吸收在肺损伤修复中具有重要作用。肺泡上皮细胞的ENaC通道负责水和电解质的重吸收，是保持肺泡液体平衡的重要离子通道。α-ENaC是ENaC的功能亚基，研究证实：全身性敲除α-ENaC基因后，新生小鼠出生后因不能有效清除肺内羊水而死亡，但对于成年小鼠的α-ENaC在肺泡中的作用研究较少。本课题利用CRISPR/Cas9技术和同源重组原理，构建scnn1aflox/-小鼠。同时通过与Sftpc::CreERT2小鼠杂交和繁育，获得了Scnn1aflox/flox和Scnn1aflox/flox/Sftpc::Cre小鼠。对Scnn1aflox/flox/Sftpc::Cre成年小鼠进行不同浓度的他莫西芬诱导，结果显示当他莫西芬浓度为100mg/Kg时，ATⅡ细胞的α-ENaC基因的敲除效率达90%以上。因此我们成功构建了ATⅡ细胞特异性敲除α-ENaC基因的成年小鼠。