Treg中条件性Notch2基因敲除小鼠的构建及鉴定

Construction and Identification of Treg-conditional Notch2-knockout Mice

目的繁育和鉴定调节性T细胞(regulatory T cells,Treg)中条件性Notch2基因敲除小鼠,为Notch2在Treg中的功能提供研究工具;为Treg与免疫性疾病的发病关系及靶向治疗提供研究手段。方法将引进的Notch2^(flox/flox)小鼠和Foxp3-Cre^(+)小鼠进行饲养和杂交;将繁育得到的第一代小鼠进行相互交配,再得到第二代小鼠。通过聚合酶链反应(polymerase chain reaction,PCR)鉴定出具有Notch2^(flox/flox)Foxp3-Cre^(+)基因型的小鼠,再通过该小鼠进行繁育。通过流式细胞术分选出野生型小鼠和Notch2^(flox/flox)Foxp3-Cre^(+)小鼠体内的Treg,Western blot检测小鼠Treg中Foxp3、Notch2、NICD2蛋白的表达,苏木精-伊红(hemotoxylin-eosin,HE)染色观察小鼠肺脏等器官的形态学变化。结果该方法成功构建了在Treg中敲除Notch2基因的小鼠。Western blot结果显示Notch2^(flox/flox)Foxp3-Cre+小鼠体内的Treg中Notch2、NICD2、Foxp3蛋白表达明显下降(P<0.05),HE染色显示该小鼠肺脏等器官出现明显炎症细胞浸润。结论本研究成功构建了Treg中条件性敲除Notch2基因的实验小鼠,为Treg与免疫性疾病的发病和靶向干预研究奠定了基础。

Objective To breed and identify conditioned Notch2 knockout mice and provide research tools for Notch2′s capabilities in regulatory T cells(Treg).To provide a research method for the pathogenesis and targeted therapy of Treg and immune diseases.Methods The introduced Notch2^(flox/flox)mice and Foxp3-Cre^(+)mice were fed and crossbred.The first-generation heterozygous mice were mated reciprocally to produce a second-generation of mice.Mice with Notch2^(flox/flox)Foxp3-Cre^(+)genotype were identified by polymerase chain reaction(PCR)and then bred these mice.Treg were isolated from wild mice and Notch2^(flox/flox)Foxp3-Cre^(+)mice by flow cytometry,and the protein expressions of Foxp3,Notch2 and NICD2 in Treg were detected by Western blot.Hemotoxylin-eosin(HE)staining was used to observe the morphological changes of lung and other organs in Notch2^(flox/flox)Foxp3-Cre^(+)mice.Results Mice with knockout of Notch2 in the Treg were successfully constructed by this method.Western blot results showed that the expressions of Notch2,NICD2,and Foxp3 in Treg of the Notch2^(flox/flox)Foxp3-Cre^(+)mice were significantly decreased(P<0.05),and HE staining displayed that the lung and other organs of the mice were obvious inflammatory cell infiltration.Conclusion In this study,experimental mice with conditional knockout of Notch2 in the Treg have been successfully constructed,laying a foundation for the study on the pathogenesis of Treg in immune diseases and its targeted intervention.