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

作　　者：吴强乐[1] 许袁琦[1] 黄宏[1] 石怡珍[1] 刘增礼[1] 

机构地区：[1]苏州大学附属第二医院核医学科,江苏苏州215004

出　　处：《苏州大学学报（医学版）》2012年第6期769-773,787,共6页Suzhou University Journal of Medical Science

基　　金：江苏省自然科学基金资助项目(BK2008164)

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>0.05)。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。Objective To establish a recombinant plasmid expressing hNIS gene regulated by pro- moter Egr-1 and then transfected to human cervical cancer cell lines Hela, to evaluate the radiation-in- duced 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 con- struct the recombinant plasmid Egr-I-hNIS/pcDNA3, and then verified by restriction enzyme electropho- resis. The recombinant plasmid was used to transfeet human cervical cancer Hela cell line with FuGENE HD. Single clone named Hela-Egr-l-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 125I dynamic uptake experiment using different doses of X-ray ionizing radiation was performed to evaluate the iodide uptake of Hela-Egr-l-hNIS and Hela-NIS （ ＋ ） cells, which was obtained from the previous study. The untransfeeted 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 af- ter 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-l-hNIS cells. 125I dynamic uptake experiments showed that the iodine uptake capacity of Hela-Egr-l-hNIS cells was increased by 13 folds than the negative control. After X-ray ionizing radiation, the iodine uptake rate of Hela-Egr-l-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-l-hNIS cell lines stably expressing the Egr-1 promoter-driven hNIS protein were established successfully, showing a strong capability of iodide uptake and a

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 

分 类 号：R394.8[医药卫生—医学遗传学]