【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。更多还原

【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 electrophoresis.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 125I 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.更多还原