关键词: 紧密连接蛋白, 基因敲除, 炎性反应, 蛋白印迹

Abstract: Objective To investigate the phenotype of Claudin-7 knockout mice (KO) and potential anti-tumor function of Claudin-7 gene. Methods DNA extracted from mouse tails were used to determine mouse genotype by PCR methods, and Claudin-7 protein expression in various tissues was detected by Western blotting, and pathomorphological changes in intestine were observed by HE staining. Interaction between protein Claudin-7 and intergrin α2 were detected by co-immunoprecipitation method.Claudin-7 protein expression in each organ and protein C-fos, C-jun, COX-2 expression in intestine of WT and KO mouse were measured by Western blotting, as well as Claudin-7 expression at different degrees of differentiation of colon cancer tissues.Results Mouse failing to grow could be found 4 days after birth for Claudin-7 knockout mice (KO), which would die within about 7 days after birth. Claudin-7 could be detected strongly in various tissues such as intestine, stomach, lung, bladder, skin, thymus and kidney, but ubiquitously expressed along the whole intestine including small and large intestine. Moreover, serious intestinal mucosal disruption including severe epithelial cell loss, vacuolation and infiltrated with inflammatory cells such as neutrophil granulocyte could be viewed by light micrographs with H&E-stained, but there were no obvious changes in other tissues except light inflammatory cells infiltration. C-fos, C-jun and Cox-2 protein expression was upregulated significantly in intestine of Claudin-7 knockout mouse as compared with that in normal mouse (WT), as well as Claudin-7 protein expression downregulated in poorly differentiated and liver metastasis tissues of colonic carcinoma. Importantly, Claudin-7 and intergrin α2 could precipitate together by co-immunoprecipitation (Co-IP) method. Conclusion Mouse intestinal inflammation model was modulated by Claudin-7 knockout successfully, and may be a potential tumor suppressor gene.

Key words: tight junction protein, gene knockout, inflammation, Western blotting