信号转导及转录活化因子基因(stat3)转染牛乳腺上皮细胞及生物学特性观察  

Bovine Mammary Epithelial Cells Transfected with Signal Transducer and Activator of Transcription 3 Gene(stat3)and Its Biological Characteristics

作　　者：刘甲[1] 何玉龙 吕品[1] 孙健红[1] 张涌[1] 

机构地区：[1]西北农林科技大学生物工程研究所,杨凌712100

出　　处：《农业生物技术学报》2009年第3期426-432,共7页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2004AA213072)资助

摘　　要：应用PCR技术从含有人信号转导及转录活化因子(stat3)基因cDNA的质粒pOTB7中扩增stat3基因片段。将其克隆入真核表达载体pEGFP-C1中,构建重组表达质粒。利用脂质体介导法将重组表达质粒转染原代牛乳腺上皮细胞(bovinemammary epithelial cells)。G418筛选阳性克隆,RT-PCR检测stat3基因在牛乳腺上皮细胞中的表达,并通过流式细胞术(flow cytometry)检测转染细胞的增殖能力、周期以及DNA含量。结果表明,转染24h后大约16%的细胞被导入含stat3基因的重组表达质粒。在mRNA水平证实细胞内有stat3基因的高表达。导入stat3基因后,细胞增殖能力增强,染色体变为三倍体,细胞的增殖寿命较对照细胞延长了13代。表明导入stat3基因能延长体外培养的牛乳腺上皮细胞的寿命。Signal transducer and activator of transcription 3 （stat3） gene was amplified from pOTB7 plasmid which contained human stat3 cDNA fragment, then inserted into pEGFP-C 1 vector to construct recombinant plasmid.The recombinant plasmid was transfected into bovine mammary epithelial （BME） cells mediated by LipofectaminTM 2000. The positive cell clones were obtained after selected by G418 and the BME cells transfected with star3 cDNA were determined by RT- PCR. Flow cytometry was used to analyze the cell cycles, multiplication capacity and DNA content. The results indicated that 24 h after exposure to the recombinant plasmid the transfection rate of the BME cells was 16%. The strong expression of star3 in transfected BME cells was confirmed at mRNA levels.After introduction of the recombinant plasmid into BME cells, the capability of proliferation was obviously enhanced and the chromosomes were changed to triploid, the cell proliferation lifespan was elongated to 13 generations doublings to control. Results indicated that the cell proliferation lifespan was elongated in vitro with introduction of the recombinant plasmid into BME cells.

关 键 词：信号转导及转录活化因子(stat3)基因 牛乳腺上皮细胞 转染 生物学特性 

分 类 号：S188[农业科学—农业基础科学]