| 李玮,谭建*,王澎,李宁,李承霞.hTERT和GFAP双启动子条件复制腺病毒介导放射性碘治疗脑胶质瘤的实验研究[J].中华放射医学与防护杂志,2014,34(1):3-7 |
| hTERT和GFAP双启动子条件复制腺病毒介导放射性碘治疗脑胶质瘤的实验研究 |
| Conditionally replicative adenovirus under the control of glial fibrillary acidic protein and human telomerase reverse transcriptase dual-promoters direct sodium iodide symporter expression for malignant glioma radioiodine therapy |
| 投稿时间：2013-05-14 |
| DOI：10.3760/cma.j.issn.0254-5098.2014.01.002 |
| 中文关键词: 条件复制腺病毒 hNIS基因 hTERT启动子 GFAP启动子 胶质瘤 |
| 英文关键词:Conditionally replicative adenovirus hNIS gene hTERT promoter GFAP promoter Glioma |
| 基金项目:国家自然科学基金（81301244，81171372）；天津市应用基础及前沿技术研究计划（12JCZDJC26000） |
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| 中文摘要: |
| 目的 利用条件复制腺病毒Ad-Tp-E1a-Gp-NIS感染脑胶质瘤细胞，探讨其介导靶向性放射性碘治疗脑胶质瘤的可行性。方法 克隆人端粒反转录酶（hTERT）和神经胶质纤维酸性蛋白（GFAP）启动子，荧光素酶分析法检测启动子启动效率。构建并纯化条件复制腺病毒Ad-Tp-E1a-Gp-NIS，进行肿瘤选择性病毒复制实验。U87和U251细胞感染Ad-Tp-E1a-Gp-NIS后，进行Western blot分析蛋白质表达，125I内流及外流和131I克隆形成实验。结果 荧光素分析实验证明，hTERT和GFAP启动子分别可以引导荧光素蛋白在U251和U87细胞中表达，表达效率为37.31%~49.00%（F=5.87，P<0.05）和59.75%~62.10%（F=11.89，P<0.01）。Ad-Tp-E1a-Gp-NIS能够在hTERT阳性和GFAP阳性实现肿瘤选择性复制，且在GFAP启动子引导下能成功表达hNIS基因。Western blot分析表明，hTERT蛋白和GFAP蛋白在U87和U251细胞中显示出相对分子质量为120×103和49×103的条带。感染Ad-Tp-E1a-Gp-NIS后，U87细胞摄碘能力提高78.80倍（F=2 914.58，P<0.01），U251细胞摄碘能力提高92.48倍（F=2 275.91，P<0.01）。体外细胞克隆分析证明，感染病毒并在131I中孵育12 h后，超过90%的肿瘤细胞被杀死，而对照组细胞仅有65%（t=11.73~78.33，P<0.01）。结论 Ad-Tp-E1a-Gp-NIS能实现条件性复制并具有明确的肿瘤靶向性。在细胞水平实验中，能引导放射性131I杀死胶质瘤细胞。 |
| 英文摘要: |
| Objective To explore the possibility of using 131I as a targeted therapy method for malignant glioma by infecting U87 and U251 cells with conditionally replicative adenovirus Ad-Tp-E1a-Gp-NIS. Methods Human telomerase reverse transcriptase (hTERT) promoter and glial fibrillary acidic protein (GFAP) promoter were cloned and their transcriptional activities were detected by luciferase assay. The conditionally replicative adenovirus Ad-Tp-E1a-Gp-NIS was constructed, purified, and transfected into U87 and U251 glioma cells. For these transfected cells, the selective replication ability was evaluated by plaque forming assay, and protein expression was detected by Western blot assay. 125I-iodide uptake and exflux, the clonoy formation of 131I-iodide treated cells were also measured. Results Transcriptions activity of the GFAP and hTERT promoters was 59.75%-62.10% (F=11.89, P<0.01) in U87 cells and 37.31%-49.00% (F=5.87, P<0.05) in U251 cells. The Ad-Tp-E1a-Gp-NIS could be selectively replicated and the hNIS gene was successfully expressed in the hTERT-positive and GFAP-positive glioma cells which showed two protein bands with relative molecular mass of 120×103 and 49×103 in Western blot assay. After infection with Ad-Tp-E1a-Gp-NIS, the cell ability of 125I uptake was increased by 78.80 (F=2 914.58, P<0.01) and 92.48 (F=2 275.91, P<0.01) times in U87 and U251 cells, respectively. The GFAP-negative MRC-5 cells could not take in 125I. The in vitro clonogenic assay indicated that, after 131I treatment, more than 90% of the transfected cells were killed, while only about 65% (t=11.73-78.33, P<0.01) of control cells were killed. Conclusions The Ad-Tp-E1a-Gp-NIS has a good ability in selective replication and the enhancement of antitumor therapy effect by increasing tumor-specific iodide uptake in malignant glioma cells. |
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