感染突变Cav-1基因的人胚肾293T细胞eNOS蛋白、NO表达变化  

Expression of eNOS protein and NO in human embryonic kidney 293T cells infected with mutant Cav-1 gene

作　　者：陈海英[1] 汪磊[1,2] 王兰花[1,2] 夏鹏[1,2] 张霄[2] 薛玉增[1] 陈双峰[1] 王乐信[1,3] 

机构地区：[1]聊城市人民医院,山东聊城252000 [2]泰山医学院聊城临床学院 [3]澳大利亚查尔斯特大学生物医学院

出　　处：《山东医药》2014年第35期5-7,共3页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81270104);山东省自然科学基金资助项目(ZR2013HL015)

摘　　要：目的观察感染突变窖蛋白-1(Cav-1)基因的人胚肾293T细胞中一氧化氮合酶(eNOS)蛋白及一氧化氮(NO)表达变化。方法采用PCR法扩增突变型Cav-1(F92A-Cav-1)基因,插入慢病毒载体pLVX-mCMV-mCherry。重组质粒转化,对阳性克隆进行测序和酶切鉴定,F92A-Cav-1成功克隆于慢病毒载体,将构建成功的重组质粒pLVX-F92A-Cav-1-mCMV-mCherry命名为LV-F92A-Cav-1;将293T细胞随机分为正常对照组、阴性对照组(空载体组)和LV-F92A-Cav-1组。正常对照组不转染,空载体组转染空载体,LV-F92A-Cav-1组转染LV-F92A-Cav-1。分别采用免疫荧光及NO荧光探针观察感染LV-F92A-Cav-1质粒的293T细胞中eNOS蛋白及NO表达,CCK-8法观察感染LV-F92A-Cav-1的293T细胞活性。结果与正常对照组及空载体组相比,LV-F92A-Cav-1组eNOS蛋白表达及NO生成增多;正常对照组、空载体组、F92A-Cav-1组293T细胞OD值分别为2.233、2.184、2.231,三组间两两比较,P均>0.05。结论感染突变Cav-1基因的人胚肾293T细胞中eNOS蛋白、NO表达增多。Objective To investigate the expression of nitric oxide synthase （ eNOS） protein and nitric oxide （ NO） in human embryonic kidney 293T cells infected with mutant Caveolin-1 （Cav-1） gene.Methods F92-Cav-1 gene was ampli-fied by PCR and inserted into the lentiviral vector backbone pLVX-mCMV-mCherry.After the recombinant plasmid trans-formation, positive clones were identified by sequencing and restriction enzyme digestion.pLVX-F92A-Cav-1-mCMV-mCherry was successfully cloned into lentiviral vector and named as F92A-Cav-1.293T cells were randomly divided into normal control group, negative control group （ empty vector group） and LV-F92A-Cav 1 group.Normal control groups were not transfected, the empty vector groups were transfected empty plasmid, LV-F92A-Cav-1 groups were transfected with LV-F92A-Cav-1.The expression of eNOS protein and NO in 293T cells infected with LV-F92A-Cav-1 plasmid were investigated by immunofluorescence and NO fluorescent probe respectively.The cell viability was measured by CCK-8.Results Com-pared with normal control group and empty vector group, the F92A-Cav-1 could increase eNOS protein expression and NO production.The Optical Density （ OD） values in the normal control groups, empty vector groups and LV-F92A-Cav-1 groups were respectively 2.233, 2.184, 2.231, pairwise comparison in three groups, all P〉0.05.Conclusion The ex-pression of eNOS protein and NO are increased in 293T cells infected with mutant Cav-1.

关 键 词：窖蛋白-1 内皮型一氧化氮合酶 一氧化氮 慢病毒载体 CAVEOLIN-1 

分 类 号：R34[医药卫生—基础医学]