Guo Rui, Zhang Ruyuan, Pan Yixin, Xu Haoping, Zhang Miao, Liang Sheng, Wang Lihua, Zhang Yifan, Li Biao
Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Nucl Med Commun. 2011 May;32(5):402-9. doi: 10.1097/MNM.0b013e328344a1ad.
Increased expression of sodium iodide symporter (NIS) is required for reporter gene imaging and effective radioiodine treatment of tumor. As the early-growth response-1 (Egr1) promoter is activated by radioisotopes, the existence of a positive feedback effect of I-promoted Egr1-hNIS expression is possible. Compared with a widely used cytomegalovirus (CMV) promoter, we investigated a possible increased activity of I-stimulated human NIS (hNIS) transgene expression in malignant glioma using a baculovirus vector containing the Egr1 promoter.
Recombinant baculovirus (Bac-CMV-hNIS) encoding the hNIS gene under the control of the CMV promoter and Bac-Egr1-hNIS encoding the hNIS gene under the control of the radiation-inducible Egrl promoter were constructed. After human malignant glioma U87 cells were transfected with Bac-CMV-hNIS or Bac-Egr1-hNIS, stimulated with or without I, the expression of the hNIS protein was detected by immunofluorescence and a flow cytometry test. The uptake and efflux of iodine were determined after the incubation of the transfected cells with I.
Immunocytochemical staining and flow cytometry test showed a lower hNIS protein expression in U87 cells transfected with Bac-Egr1-hNIS (even after I stimulation) compared with U87 cells transfected with Bac-CMV-hNIS. Bac-CMV-hNIS-transfected U87 cells accumulated up to approximately 25.8 times more I than nontransfected cells, whereas Bac-Egr1-hNIS-transfected U87 cells accumulated up to approximately 3.5 and 14.2 times more I pre-stimulation and post-stimulation. However, rapid efflux of radioactivity was observed in both groups, with 50% lost during the first 2 min after the I-containing medium was replaced by a nonradioactive medium.
Our results indicated that an improved transgene expression of I-stimulated hNIS in U87-malignant glioma cells using a baculovirus vector containing the Egr1 promoter is possible, but the expression level is lower than that of Bac-CMV-hNIS-transfected U87 cells. However, it might be an approach to improve the specificity of gene therapy using radiosensitive promoters to activate hNIS gene expression selectively in the radiation field.
报告基因成像及肿瘤的有效放射性碘治疗需要增加碘化钠同向转运体（NIS）的表达。由于早期生长反应因子1（Egr1）启动子可被放射性同位素激活，因此碘促进Egr1-hNIS表达存在正反馈效应。与广泛使用的巨细胞病毒（CMV）启动子相比，我们使用含有Egr1启动子的杆状病毒载体，研究了在恶性胶质瘤中碘刺激的人NIS（hNIS）转基因表达活性可能的增加情况。
构建在CMV启动子控制下编码hNIS基因的重组杆状病毒（Bac-CMV-hNIS）和在辐射诱导性Egrl启动子控制下编码hNIS基因的Bac-Egr1-hNIS。用Bac-CMV-hNIS或Bac-Egr1-hNIS转染人恶性胶质瘤U87细胞，在有无碘刺激的情况下，通过免疫荧光和流式细胞术检测hNIS蛋白的表达。在用碘孵育转染细胞后，测定碘的摄取和流出情况。
免疫细胞化学染色和流式细胞术检测显示，与用Bac-CMV-hNIS转染的U87细胞相比，用Bac-Egr1-hNIS转染的U87细胞（即使在碘刺激后）中hNIS蛋白表达较低。用Bac-CMV-hNIS转染的U87细胞积累的碘比未转染细胞多约25.8倍，而用Bac-Egr1-hNIS转染的U87细胞在刺激前和刺激后积累的碘分别多约3.5倍和14.2倍。然而，两组均观察到放射性的快速流出，在用含碘培养基被无放射性培养基替换后的前2分钟内，50%的放射性丧失。
我们的结果表明，使用含有Egr1启动子的杆状病毒载体在U87恶性胶质瘤细胞中提高碘刺激的hNIS转基因表达是可能的，但表达水平低于用Bac-CMV-hNIS转染的U87细胞。然而这可能是一种利用放射敏感启动子在辐射场中选择性激活hNIS基因表达来提高基因治疗特异性的方法。
