巨噬细胞条件性Cd226基因敲除小鼠的构建及功能分析

Construction and functional analysis of macrophage-conditional Cd226 gene knockout mice

目的构建并鉴定巨噬细胞条件性Cd226基因敲除小鼠,为研究CD226分子调节巨噬细胞表型和功能影响疾病发生发展提供动物模型。方法通过Cd226^(flox/+)雌雄小鼠自交,得到基因型Cd226^(flox/flox)小鼠。Cd226^(flox/flox)与Lyz2-Cre^(+)小鼠杂交,获得Cd226^(flox/+)Lyz2-Cre^(+)小鼠,再将其与Cd226^(flox/flox)小鼠杂交,最终获得Cd226^(flox/flox)Lyz2-Cre^(+)小鼠,即巨噬细胞条件性Cd226基因敲除小鼠。采用PCR和琼脂糖凝胶电泳鉴定小鼠基因型;采用流式细胞术、qRT-PCR和Western Blot验证小鼠巨噬细胞CD226敲除效果;采用Transwell实验检测CD226对小鼠巨噬细胞迁移能力的影响。结果从基因和蛋白水平证实巨噬细胞条件性Cd226基因敲除小鼠构建成功。Transwell实验结果显示,与对照组相比,巨噬细胞条件性敲除CD226分子显著抑制腹腔巨噬细胞的迁移。结论成功构建巨噬细胞条件性Cd226基因敲除小鼠,为后续深入研究CD226调控巨噬细胞功能在免疫性疾病发病中的作用和机制提供更加精准的实验动物模型。

Objective To construct and identify macrophage-conditional Cd226 gene knockout mice,and to provide an animal model for studying the effect of CD226 on the occurrence and development of diseases by regulating the phenotype and function of macrophages.Methods Cd226^(flox/+)mice were self-crossed by male and female to obtain Cd226^(flox/flox)mice.Cd226^(flox/flox)were hybridized with Lyz2-Cre^(+)mice to obtain Cd226^(flox/+)Lyz2-Cre^(+)mice,and then crossed with Cd226^(flox/flox)mice to obtain macrophage-conditional Cd226 knockout mice(Cd226^(flox/flox)Lyz2-Cre^(+)).The phenotypes of the mice were identified by PCR and agarose gel electrophoresis.To verify that CD226 had been conditionally knockdown in macrophages,qRT-PCR,flow cytometry and Western Blot were used to evaluate CD226 expression.Transwell was used to dectect the effect of CD226 on the migration of macrophages.Results The successful establishment of macrophage-conditional Cd226 gene knockout mice was confirmed at gene and protein levels.Compared with Cd226^(flox/flox)mice,the migration ability of peritoneal macrophages was significantly inhibited in Cd226^(flox/flox)Lyz2-Cre^(+)mice.Conclusions Macrophage-conditional Cd226 gene knockout mice were successfully established,which can provide a more accurate animal model for studying the role and mechanism of CD226 regulation of macrophage in the pathogenesis of immune diseases.