一种高效稳定的磷酸钙转染293T细胞方法的建立及评价  

Establishment and evaluation of a highly efficient and stable calcium phosphate transfection method for 293T cells

作　　者：华进[1] 程志彬 林春霖 戴起宝[1] 朱广伟[1,2] HUA Jin;CHENG Zhibin;LIN Chunlin;DAI Qibao;ZHU Guangwei(Department of Gastrointestinal Surgery 2 Section, First Affiliated Hospital, Fujian Medical University,Fuzhou 350005, China;Key Laboratory of Gastrointestinal Cancer, Ministry of Education, Fujian MedicalUniversity, Fuzhou 350005 China)

机构地区：[1]福建医科大学附属第一医院胃肠外科二区,福建福州350005 [2]福建医科大学消化道恶性肿瘤教育部重点实验室,福建福州350005

出　　处：《吉林大学学报（医学版）》2019年第5期1177-1181,I0006,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81702424,81872364);国家卫健委临床重点专科建设项目(普通外科)资助课题(2013);福建省科技厅自然科学基金资助课题(2018J05127);福建省卫健委中青年骨干项目资助课题(2018-ZQN-46);福建省教育厅中青年教师教育科研项目资助课题(JAT170239)

摘　　要：目的:探讨2种磷酸钙转染法转染293T细胞的效果,建立一种实现高效稳定磷酸钙转染293T细胞的方法。方法:荧光显微镜观察采用2种磷酸钙转染方法(传统磷酸钙转染法和改良磷酸钙转染法)转染pCDH-GFP-3xflag-TRAF6质粒进入293T细胞的转染效率。采用Real-time PCR法和Western blotting法分别检测2种磷酸钙转染方法转染pCDH-GFP-3xflag-TRAF6质粒进入293T细胞后,肿瘤坏死因子受体相关因子6(TRAF6) mRNA和flag蛋白表达水平。结果:荧光显微镜下观察,与传统磷酸钙转染法比较,改良磷酸钙转染法转染pCDH-GFP-3xflag-TRAF6质粒进入293T细胞24 和48 h后的转染效率明显升高( P <0.01);Real-time PCR法和Western blotting法检测,与传统转染方法比较,磷酸钙转染改良法转染pCDH-GFP-3xflag-TRAF6质粒进入293T细胞24 和48 h后,TRAF6 mRNA 和flag蛋白表达水平均明显升高( P <0.01)。结论:本研究建立的改良磷酸钙转染方法是一种高效、稳定的DNA转染方法。Objective : To explore the effects of two kinds of calcium phosphate transfection methods in the 293T cells, and to establish the method of achieving high-efficiency and stable calcium phosphate transfection in the 293T cells. Methods : Fluorescence microscope was used to observe the transfection efficiencies of transfection of pCDH-GFP-3xflag-TRAF6 plasmid into the 293T cells by two kinds of calcium phosphate transfection methods (traditional calcium phosphate transfection method and improved calcium phosphate transfection method). Real-time PCR and Western blotting method were used to detect the expression levels of TRAF6 mRNA and flag protein in the 293T cells after transfection of pCDH-GFP-3xflag-TRAF6 plasmid by two kinds of calcium phosphate transfection methods. Results : Under fluorescence microscope, compared with traditional calcium phosphate transfection method, the transfection efficiencies of improved calcium phosphate transfection method 24 an 48 h after transfection of pCDH-GFP-3xflag-TRAF6 p lasmid into the 293T cells were significantly increased ( P <0.01). The Real-time PCR and Western blotting results showed that compared with traditional transfection method, the expression levels of TRAF6 mRNA and flag protein in the 293T cells 24 and 48 h after transfection of pCDH-GFP-3xflag-TRAF6 plasmid by calcicum phosphate transfection method were significantly increased( P <0.01). Conclusion : The improved calcium phosphate transfection method established in this reseach is a highly efficient and stable DNA transfection method.

关 键 词：磷酸钙转染 293T细胞 肿瘤坏死因子受体相关因子6 flag蛋白 

分 类 号：Q819[生物学—生物工程]