人转铁蛋白受体基因载体的构建与鉴定及在Hela细胞中的表达  

Construction and identification of hTfR gene vector and expression in Hela cells

作　　者：张斌[1] 杨卉[1] 肖煜东[1] 秦燕[1] 周顺科[1] 

机构地区：[1]中南大学湘雅二医院放射科,长沙410011

出　　处：《中华消化病与影像杂志（电子版）》2013年第2期28-33,共6页Chinese Journal of Digestion and Medical Imageology(Electronic Edition)

基　　金：湖南省科技计划项目(06FJ3167)

摘　　要：目的运用人转铁蛋白受体(hTfR)作为MR报告基因,制作包含hTfR蛋白编码区的表达质粒,并对其进行检测鉴定,为下一步体外、体内MR分子影像研究打下基础。方法通过脂质体介导的基因转染构建过量表达hTfR的Hela细胞系,以pCDNA3.1(+)-hTfR为实验组,转染空白质粒pCDNA3.1(+)为对照组,运用PCR及Westen-Blot的方法在基因水平和蛋白水平检测hTfR在转染前后的表达水平,鉴定转染效果。结果以pOTB7-TFRC质粒为模板,运用RT-PCR的方法进行hTfR基因编码区的扩展,成功构建了pCDNA3.1(+)-hTfR高表达质粒,测序鉴定正确;通过脂质体转染Hela细胞,瞬时转染效率约70%;RT-PCR显示转染pCDNA3.1(+)-hTfR质粒组较对照组生成的hTfR mRNA明显增高(1.179±0.047 vs.0.260,t=16.8,P>0.05);Western-Bolt显示转染组较对照组编码的TfR蛋白明显增高(0.648±0.025 vs.0.333,t=10.8,P<0.05)。结论成功构建了pCDNA3.1(+)-hTfR表达质粒;质粒转染Hela细胞后表达的hTfR mRNA及hTfR蛋白均明显增高;为下一步分子影像学的实验奠定了基础。Objective To construct and identify pCDNA3. 1 （ ＋ ）-hTfR expression plasmid for further molecular imaging research. Methods Hela cells line with high expression of hTfR was established via liposome mediated transfection, pCDNA3. 1 （ ＋ ）-hTfR transfected Hela cells were used as the test group, and empty vector-transfected Hela cells were used as the control group. The mRNA and protein expressions of hTfR were detected with PCR and Westen-Blot before and after transfection, and the efficiency of transfection was identificated. Results pOTB7-TFRC plasmid as a template, the coding region of hTfR gene was amplified through reverse transcription-polymerase chain reaction （RT-PCR） , pCDNA3.1 （ ＋ ）- hTfR over-expression plasmid was constructed successfully, and the sequence was correct; the efficiency of transient transfection to Hela cells by liposome was about 70% ; RT-PCR showed that hTfR mRNA in test group was significantly higher than that in control group （ 1. 179 ±0. 047 vs. 0. 260, t = 16.8, P 〈 0.05 ） ; Western-Bolt showed that the expression of hTfR protein in test group was significantly higher than that in control group （0. 648 ±0. 025 vs. 0. 333, t = 10. 8, P 〈 0.05 ）. Conclusions pCDNA3. 1 （ ＋ ）-hTfR expression plasmid can be constructed successfully; hTfR mRNA and hTfR protein increase strongly after transient transfection ; this study establishes infarctate foundation for empirical research of molecular imaging of MR.

关 键 词：HELA细胞 受体 转铁蛋白 质粒 逆转录聚合酶链反应 分子影像学 

分 类 号：R392[医药卫生—免疫学]