Egr-1启动子调控hNIS基因的重组质粒构建及转染细胞株的建立
The construction of recombinant plasmid expressing hNIS gene regulated by promoter Egr-1 and establishment of its stable transfected cell lines
吴强乐 1许袁琦 1黄宏 1石怡珍 1刘增礼1
作者信息
- 1. 苏州大学附属第二医院核医学科,江苏苏州215004
- 折叠
摘要
目的 构建早期生长反应基因-1(Egr-1)启动子调控人钠碘转运体(hNIS)基因的重组质粒并稳定转染人宫颈癌细胞株Hela,评价Egr-1启动子对hNIS蛋白功能的辐射诱导作用.方法 以Egr-1/pMR18T为模板PCR扩增Egr-1启动子序列,亚克隆至真核表达载体FL*-hNIS/pcDNA3,构成重组质粒Egr-1-hNIS/pcDNA3,酶切电泳鉴定,通过FuGENE HD转染入Hela细胞,G418筛选抗性克隆,获取单克隆Hela-Egr-1-hNIS.分别采用流式细胞仪、RT-PCR检测Hela-Egr-1-hNIS细胞系中NIS基因和NIS蛋白的表达.动态摄取碘-125实验观察Hela-Egr-1-hNIS细胞和前期研究所得细胞株Hela-NIS(+)给予不同剂量的X线辐照前后的摄碘功能.未转染的Hela细胞作为阴性对照组.结果 成功构建重组质粒Egr-1-hNIS/pcDNA3,转染Hela细胞后获得了稳定表达细胞系Hela-Egr-1-hNIS,流式细胞仪测得NIS表达效率为11.2％；RT-PCR检测到Hela-Egr-1-hNIS有NIS mRNA的转录,而对照组无NIS蛋白表达.动态摄碘实验提示Hela-Egr-1-hNIS细胞摄碘能力比阴性对照Hela细胞提高了13倍；辐照后摄碘能力增强,在0～6 Gy范围内与辐射剂量成正相关(r=0.960,P＜0.05)；而Hela-NIS(+)组辐照后摄碘功能下降,与辐射剂量无相关性(r=-0.770,P＞O.05).结论 成功构建Egr-1启动子调控的稳定表达hNIS蛋白的Hela-Egr-1-hNIS细胞系,该细胞系具有较强的摄碘功能及辐射诱导作用,有望在体内治疗中获得更好的治疗效果.
Abstract
Objective To establish a recombinant plasmid expressing hNIS gene regulated by promoter Egr-1 and then transfected to human cervical cancer cell lines Hela, to evaluate the radiation-induced Egr-1 promoter of hNIS protein function. Methods Egr-1 promoter sequence was amplified by PCR from the template Egr-1/pMR18T and was sub-cloned into the vector FL * -hNIS/pcDNA3, to construct the recombinant plasmid Egr-1 -hNIS/pcDNA3, and then verified by restriction enzyme electropho-resis. The recombinant plasmid was used to transfect human cervical cancer Hela cell line with FuGENE HD. Single clone named Hela-Egr-1-hNIS was obtained by G418 screen and expanded in selection media. The expression of NIS gene was detected by flow cytometry (FCM) and RT-PCR, respectively. A 125 I dynamic uptake experiment using different doses of X-ray ionizing radiation was performed to evaluate the iodide uptake of Hela-Egr-1 -hNIS and Hela-NIS ( + ) cells, which was obtained from the previous study. The untransfected Hela cells were used as the control. Results The recombinant plasmid Egr-1- hNIS/pcDNA3 was established successfully and the cell lines stably expressing NIS gene were obtained after transfecting into Hela cells. The highest level of expression efficiency of NIS was 11. 2% with FCM, and the expression of NIS gene was proved by RT-PCR from Hela-Egr-1-hNIS cells. 125I dynamic uptake experiments showed that the iodine uptake capacity of Hela-Egr-1-hNIS cells was increased by 13 folds than the negative control. After X-ray ionizing radiation, the iodine uptake rate of Hela-Egr-1-hNIS cells was increased and have a positive correlation with the radiation dose from zero to six Gy( r =0. 960,P = 0.04) , while the uptake rate of Hela-NIS( + ) was decreased and had no correlation with the dose of X-ray( r= -0. 770,P >0. 05). Conclusion The Hela-Egr-1-hNIS cell lines stably expressing the Egr-1 promoter-driven hNIS protein were established successfully, showing a strong capability of iodide uptake and a certain effect of radiation inducement, and may achieve a better therapeutic effect in vivo.
关键词
早期生长反应基因-1/钠碘转运体/宫颈肿瘤/放射性核素Key words
Egr-1/sodium-iodide symporter/uterine cervical neoplasms/iodine radioisotopes引用本文复制引用
出版年
2012
NETL
NSTL
维普
万方数据