巨噬细胞特异性TDO2基因敲除小鼠的构建  

Construction of macrophage-specific TDO2 knockout mice

作　　者：董伟波 陈悦兰 王燚 程梦 魏伟[1] 常艳[1] Dong Weibo;Chen Yuelan;Wang Yi;Cheng Meng;Wei Wei;Chang Yan(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-Inflammatory and Immune Medicine,Ministry of Education,Center of Rheumatoid Arthritis of Anhui Medical University,Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所,抗炎免疫药物教育部重点实验室,安徽省抗炎免疫药物协同创新中心,安徽医科大学类风湿关节炎研究中心,合肥230032

出　　处：《安徽医科大学学报》2024年第6期994-1000,共7页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金面上项目(编号:2108085MH320);省留学人员创新项目择优资助计划项目(编号:2022LCX019)。

摘　　要：目的构建巨噬细胞特异性色氨酸2,3-双加氧酶(TDO2)基因敲除小鼠,为研究TDO2调控巨噬细胞功能影响疾病发生发展提供动物模型。方法基于Cre-Loxp系统构建巨噬细胞特异性TDO2基因敲除(TDO2^(flox/flox)Lyz2-iCre^(+))小鼠。采用PCR和琼脂糖凝胶电泳鉴定小鼠基因型;采用Western blot和免疫荧光验证小鼠巨噬细胞中TDO2敲除效果;通过苏木精-伊红(HE)染色,观察主要组织器官是否存在自发病变。结果基因型鉴定结果表明,flox扩增产物长度在407 bp或408 bp处仅有一条条带且Cre扩增产物长度在543 bp处有一条条带的小鼠即为TDO2^(flox/flox)Lyz2-iCre^(+)小鼠。Western blot结果显示,与TDO2^(flox/flox)小鼠相比,TDO2^(flox/flox)Lyz2-iCre^(+)小鼠骨髓来源的巨噬细胞(BMDMs)中TDO2表达降低(P<0.01)。免疫荧光结果显示,与TDO2^(flox/flox)小鼠相比,TDO2^(flox/flox)Lyz2-iCre^(+)小鼠腹腔巨噬细胞和BMDMs中TDO2表达降低。HE染色结果显示,与TDO2^(flox/flox)小鼠相比,TDO2^(flox/flox)Lyz2-iCre^(+)小鼠肝脏、脑、肾脏和脾脏组织内细胞形态无明显差异。结论成功构建TDO2^(flox/flox)Lyz2-iCre^(+)小鼠,为后续深入研究TDO2调控巨噬细胞活化在疾病中的作用和机制提供更加精准的实验动物模型。Objective To provide an animal model for studying the effect of TDO2 on the function of macrophages on the occurrence and development of diseases by constructing macrophage-specific tryptophan,2,3-dioxygenase(TDO2)gene knockout mice.Methods TDO2^(flox/flox) Lyz2-iCre^(+) mice were constructed based on Cre/LoxP system.The genotypes of mice were identified by PCR amplification and agarose gel electrophoresis.Western blot and immunofluorescence were used to verify the effect of TDO2 knockdown in mouse macrophages.The spontaneous lesions in major tissues and organ were observed by HE stainings.Results The results of genotype identification showed that the mice with only one band at 407 bp or 408 bp for the flox amplification product and one band at 543 bp for the Cre amplification product were TDO2^(flox/flox) Lyz2-iCre^(+)mice.Western blot results showed that TDO2 expression in bone marrow-derived macrophages(BMDMs)of TDO2^(flox/flox) Lyz2-iCre^(+)mice decreased compared with TDO2^(flox/flox) mice(P<0.01).Immunofluorescence results showed that TDO2 expression in peritoneal macrophages and BMDMs of TDO2^(flox/flox) Lyz2-iCre^(+) mice decreased compared with TDO2^(flox/flox) mice.HE staining showed no significant differences in cell morphology in the liver,brain,kidney and spleen tissues of TDO2^(flox/flox) Lyz2-iCre^(+)mice compared to TDO2^(flox/flox) mice.Conclusion TDO2^(flox/flox) Lyz2-iCre^(+)mice is successfully constructed,providing a more precise experimental animal model for subsequent in-depth study of the role and mechanism of TDO2-regulated macrophage activation in disease.

关 键 词：TDO2 特异性基因敲除 巨噬细胞 基因型鉴定 CRE-LOXP 

分 类 号：R332[医药卫生—人体生理学]