目的 Aqp5蛋白的C末端结构对其功能的发挥起着重要作用.本研究旨在通过构建Aqp5-C末端6个氨基酸缺失的突变小鼠,验证Aqp5-C末端6个氨基酸缺失对该蛋白亚细胞定位的影响,并初步比较研究此突变小鼠的肺脏组织结构和转录组的改变,探讨该突变小鼠用于Aqp5功能研究的潜在应用价值.方法 采用基于gRNA的CRISPER Cas 9技术构建Aqp5末端6个氨基酸缺失的突变小鼠(Aqp5-p.I260*)并进行序列鉴定,采用组织切片、荧光染色和显微镜观察等手段明确突变蛋白在肺泡上皮细胞的亚细胞定位和突变小鼠肺组织结构,采用转录组测序研究突变小鼠肺脏基因差异表达.结果 本研究成功构建了Aqp5蛋白C末端截断小鼠,发现p.I260*改变了亚细胞定位,Aqp5-p.I260*鼠肺泡数目少于野生鼠;Aqp5-p.I260*小鼠肺组织的转录组上调基因主要富集于发育和造血细胞谱系等通路,其Aqp5功能缺陷后诱发炎症相关通路基因(Hspa1a、IL-1β、Col3a1等)的表达水平改变.结论 本研究在体验证了小鼠Aqp5蛋白C末端6个氨基酸缺失突变对该蛋白细胞膜定位的关键作用,表明了以该小鼠作为Aqp5功能缺陷模型探讨Aqp5蛋白在肺发育和骨髓造血、炎症和免疫调节等生物学功能的潜在应用价值.
The Construction of Mice Model with C-terminal Truncated Aqp5 and Its Potential Applications
Objective The C-terminal amino acids of Aqp5 protein may play an important role in its general function.In this study,we constructed a mouse model with a C-terminal truncation of the Aqp5 protein to verify the effect of C-terminal amino acids on the subcellular localization of Aqp5 protein.Additionally,we conducted a preliminary analysis comparing alterations in lung structure and transcriptome between wildtype mice and this mutant model.Further investigation was conducted to explore the potential application value of this mouse as an Aqp5 loss-of-function model for its functional study.Methods gRNA-based CRISPER Cas 9 knock-in was applied to construct the Aqp5 mutant mice model and PCR -based sequences were used for validation.The subcellular localization of the mutant Aqp5 protein in alveolar epithelial cells and the lung structure were investigated by histological analysis,followed by fluorescence staining and microscopic observation.Transcriptome sequencing was used to study the differential gene expression in the lung structure of mutant mice.Results Aqp5 C-terminal deletion mice were successfully generated and we found that Aqp5-p.I260* mice changed the subcellular localization.The number of alveoli in Aqp5-p. I260* mice was significantly less than that in the wild type mice.The differentially expressed genes in the lung tissues of Aqp5-p.I260* mice were mainly involved in development and hematopoietic lineage pathways.Aqp5 deficiency in the lung tissues induced changes in the expression levels of inflation-related genes (Hspa1a,IL-1 betas,Col3a1 )in the lung tissues.Conclusion The significance of the C-terminal 6 amino acids in Aqp5 for its localization to the cell membrane was validated through the construction of a mutant mouse model.This preliminary study indicates that the potential application value of Aqp5 as a model for Aqp5 deficiency in the future study of the biological functions for Aqp5 in lung development,bone marrow hematopoiesis,inflammation and immune regulation.
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