巨噬细胞特异性敲除一磷酸腺苷激活依赖性蛋白激酶基因的小鼠模型构建  

Construction of macrophage-specific Adenosine 5′-monophosphate-activated progein kinase knockout mouse model

作　　者：郑帅[1] 刘燕[1] 朴春梅[1] 刘婷婷[1] 王吉静 王绿娅[2] 杜杰[1] ZHENG Shuai;LIU Yan;PIAO Chunmei;LIU Tingting;WANG Jijing;WANG Lvya;DU Jie(Department of Cardiovascular Biology,Beijing Anzhen Hospital,Capital Medical University and Beijing Institute of Heart Lung and Blood Vessel Diseases,Beijing 100029,China)

机构地区：[1]首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所心血管生物研究室心血管重塑相关疾病教育部重点实验室北京市心血管重大疾病协同创新中心,100029 [2]首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所动脉粥样硬化研究室,100029 [3]河南省人民医院&郑州大学人民医院心功能科

出　　处：《心肺血管病杂志》2019年第7期788-792,共5页Journal of Cardiovascular and Pulmonary Diseases

基　　金：国家自然科学基金项目(81400194)

摘　　要：目的:构建巨噬细胞特异性敲除一磷酸腺苷激活依赖性蛋白激酶(AMPK)基因的小鼠模型,并观察对血压的影响。方法:利用胚胎显微注射法构建AMPKα1基因第3外显子两端携带loxP位点的小鼠模型,和集落刺激因子1受体启动子诱导表达Cre酶的小鼠模型,交配繁育出巨噬细胞特异性敲除AMPK的小鼠模型。鼠尾DNA做PCR鉴定基因型,腹腔注射他莫昔芬诱导敲除AMPK,Real-timePCR检测小鼠骨髓细胞AMPKRNA水平。检测Cre阴性小鼠注射与不注射他莫昔芬的血压差异,以及Cre阴性和阳性小鼠注射他莫昔芬5d后的血压差异。结果:鼠尾DNAPCR鉴定出AMPKα1loxP为纯合阳性、且Cre为阴性或阳性的小鼠。与Cre阴性小鼠相比,他莫昔芬显著降低了Cre阳性小鼠骨髓细胞AMPKmRNA[(0.9263±0.1293)vs.(0.47±0.04657),P<0.017]。他莫昔芬对Cre阴性小鼠血压的影响不显著[收缩压(122.9±3.183)vs.(124±6.878)mmHg,1mmHg=0.133kPa,P=0.427],而AMPK敲除鼠血压显著低于对照组[收缩压(123.5±3.577)vs.(107.5±4.814)mmHg,P=0.037]。结论:成功构建巨噬细胞特异性敲除AMPK的小鼠模型,证实敲除鼠血压降低。Objective: To breed a macrophage-specific AMP-activated protein kinase ( AMPK) knockout mouse model,and to observe the influence of such knockout on blood pressure. Methods: Using Embryo microinjection method to construct transgenic mice which had the loxP sites inserted in both upstream and downstream of exon3 of AMPKα1 gene,meanwhile construct transgenic mice which express Cre recombinase / mutant murine estrogen receptor double-fusion protein under the direction of the mouse Csf1r ( colony stimulating factor 1 receptor) promoter. Cross breeding the two transgenic mice to generate tamoxifen-inducible macrophage-specific AMPK knockout mice. The genotype of AMPK conditional knockout mice was analyzed by PCR test on mice tail DNA samples. AMPK knockout was induced by intraperitoneal injection of tamoxifen. To verify the knockout efficiency,bone marrow cells RNA from both Cre-negative and Cre-positive mice were extracted after tamoxifen injection,and had AMPKα1 level evaluated by real-time PCR. Blood pressure were compared between Cre-negative mice either had tamoxifen injection or not,and between Cre-negetive and Cre-positive mice after tamoxifen injection. Results: Genotyping by mice tails DNA PCR could identify AMPKα1 loxP homozygous positive,meanwhile with either Cre-negative or Cre-positive genotype mice. Compared with Crenegative mice,tamoxifen injection significantly downregulated AMPKα1 mRNA levels in Cre-positive mice [( 0. 9263±0. 1293) vs.( 0. 47±0. 04657),P <0. 017]. The blood pressure showed little difference between no tamoxifen injection group and tamoxifen injection group in Cre-negative mice[SBP( 122. 9±3. 183) vs.( 124± 6. 878) mmHg,P= 0. 427]. While the blood pressure was significantly lower in tamoxifen-induced AMPKα1 knockout mice ( Cre-positive) than in the non-knockout mice ( Cre-negative)[SBP was( 123. 5± 3. 577) vs.( 107. 5 ± 4. 814) mmHg,P = 0. 037]. Conclusions: The macrophage-specific tamoxifen-inducible AMPK knockout mouse model was successfully constructed,and proved downreg

关 键 词：巨噬细胞 一磷酸腺苷激活依赖性蛋白激酶 组织特异性敲除 小鼠模型 

分 类 号：R54[医药卫生—心血管疾病]