GFAP及hTERT双启动子靶向性表达hNIS基因引导放射性碘治疗胶质瘤
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摘要
研究目的放射性碘治疗甲状腺癌是临床常用方法。非甲状腺来源肿瘤通过转染hNIS基因也能摄取碘,端粒酶(human telomerase reverse transcriptase, hTERT)和神经胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)启动子都是肿瘤特异性启动子,对于靶向性治疗胶质瘤有潜在的研究价值。
方法先扩增GFAP、hTERT启动子等基因,并保存于相应质粒中。并测定各启动子的启动效率,然后构建了hTERT启动子引导腺病毒早起蛋白(early region1A, E1A)基因同时GFAP启动子引导钠碘转运体(human sodium iodide symporter, hNIS)基因的条件复制腺病毒。胶质瘤细胞被腺病毒转染后,利用病毒空斑形成实验检测条件复制腺病毒的复制能力。进行Western blots实验,1251内流及外流实验并进行1311细胞克隆实验,来评估细胞摄碘情况。最后,在细胞实验的基础上,构建胶质瘤荷瘤裸鼠模型,进行动物体内放射性碘治疗实验。结果成功扩增了相应基因并保存。实验证明GFAP启动子启动效率能达到SV40启动子的60%左右,hTERT启动子启动效率能达到SV40启动子的43%左右。成功构建了条件复制腺病毒Ad-Tp-E1A-Gp-NIS,并感染胶质瘤细胞。Western blot实验显示43、70、49、120、70、110kDa的条带,分别对应着β-acting、GFAP、 hTERT、hNIS和hTPO蛋白。病毒复制实验证明了Ad-Tp-E1A-Gp-NIS的有效性,hTERT启动子能成功的限制腺病毒在肿瘤细胞内的复制。摄1251实验证明GFAP启动子能成功引导hNIS基因表达并摄取碘。细胞克隆实验中显示感染Ad-Tp-E1A-Gp-NIS后肿瘤细胞能成功的被1311杀死。成功构建了U87荷瘤裸鼠模型了,感染Ad-Tp-E1A-Gp-NIS并进行放射性碘治疗后裸鼠生存周期延长,肿瘤生长被抑制,核素显像显示荷瘤裸鼠肿瘤部位能靶向性摄取放射性核素。
结论实验证明感染条件复制腺病毒Ad-Tp-E1A-Gp-NIS后,细胞实验及动物实验均证明,胶质瘤细胞系表现出肿瘤特异性的碘摄取,并引导针对胶质瘤的放射性碘治疗。
Objective Radioiodine is a routine therapy for differentiated thyroid cancers. The non-thyroid cancers can be treated with radioiodine following transfection with the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter and the glial fibrillary acidic protein(GFAP) promoter are the effective tumor-specific promoter of gene expression and thus may be useful in targeted gene therapy of Malignant glioma.
Methods The hTERT promoter, the adenoviral early region1A (E1A),the GFAP promoter and the NIS gene were amplified by PCR or DNA replication. Detected the transcriptional activity of the hTERT and GFAP gene promoter. The GFAP promoter and hNIS gene segments,the hTERT promoter and E1A gene were ligated, respectively,and package the recombinant adenovirus Ad-Tp-E1A-Gp-NIS. The U87and U251glioma cells was tranfected by Ad-Tp-ElA-Gp-NIS, then the selective replication ability of the conditionally replicative adenovirus was evaluated by plaque forming assay, and proceeded western blots,125I uptake and exflux, clonogenecity following131I treatment.At last, on the basis of the cells-level experimental data, we had established U87tumor xenografts in nude mice and proceeded radioiodine therapy in U87tumors nude mice.
Results Genes had cloned into plasmids. The activity of GFAP promoter and hTERT promoter had reached to as much as about60%and43%of SV40promoter,respectively. The recombined adenovirus Ad-Tp-E1A-Gp-NIS was constructed successfully and tranfected the glioma cells. The western blots revealed bands of approximately43,70,120,49,110kDa, consistent with the β-acting, GFAP, hTERT,hNIS and hTPO proteins. The adenovirus selective-eplication experiment was proved the efficiency of Ad-Tp-E1A-Gp-NIS and the hTERT promoter could controled the eplication of Ad-Tp-E1A-Gp-NIS in the cell-lines. After transfection with the hNIS gene, the cells could intake125I-iodide. In clonogenic assay, transfected cells were killed, compared to control cells (transfected with Ad-CMV-EGFP) after incubated with131I. The U87tumor nude mice was evaluated. After treatment with131I and injection of Ad-Tp-E1A-Gp-NIS, the survival period of U87tumor loading nude mice were prolonged and the growth of tumor in mice was inhibited. Rude mice harboring xenografts after injection of Ad-Tp-E1A-Gp-NIS, could taken the99mTc.
Conclusions The experiments demonstrated that after transfected replication-selective adeno viruses Ad-Tp-E1A-Gp-NIS, the therapy of131I was achieved effectively in malignant glioma cell lines following induction of tumor-specific iodide uptake activity by GFAP promoters directed hNIS gene expression in vitro and in vivo.
方法先扩增GFAP、hTERT启动子等基因,并保存于相应质粒中。并测定各启动子的启动效率,然后构建了hTERT启动子引导腺病毒早起蛋白(early region1A, E1A)基因同时GFAP启动子引导钠碘转运体(human sodium iodide symporter, hNIS)基因的条件复制腺病毒。胶质瘤细胞被腺病毒转染后,利用病毒空斑形成实验检测条件复制腺病毒的复制能力。进行Western blots实验,1251内流及外流实验并进行1311细胞克隆实验,来评估细胞摄碘情况。最后,在细胞实验的基础上,构建胶质瘤荷瘤裸鼠模型,进行动物体内放射性碘治疗实验。结果成功扩增了相应基因并保存。实验证明GFAP启动子启动效率能达到SV40启动子的60%左右,hTERT启动子启动效率能达到SV40启动子的43%左右。成功构建了条件复制腺病毒Ad-Tp-E1A-Gp-NIS,并感染胶质瘤细胞。Western blot实验显示43、70、49、120、70、110kDa的条带,分别对应着β-acting、GFAP、 hTERT、hNIS和hTPO蛋白。病毒复制实验证明了Ad-Tp-E1A-Gp-NIS的有效性,hTERT启动子能成功的限制腺病毒在肿瘤细胞内的复制。摄1251实验证明GFAP启动子能成功引导hNIS基因表达并摄取碘。细胞克隆实验中显示感染Ad-Tp-E1A-Gp-NIS后肿瘤细胞能成功的被1311杀死。成功构建了U87荷瘤裸鼠模型了,感染Ad-Tp-E1A-Gp-NIS并进行放射性碘治疗后裸鼠生存周期延长,肿瘤生长被抑制,核素显像显示荷瘤裸鼠肿瘤部位能靶向性摄取放射性核素。
结论实验证明感染条件复制腺病毒Ad-Tp-E1A-Gp-NIS后,细胞实验及动物实验均证明,胶质瘤细胞系表现出肿瘤特异性的碘摄取,并引导针对胶质瘤的放射性碘治疗。
Objective Radioiodine is a routine therapy for differentiated thyroid cancers. The non-thyroid cancers can be treated with radioiodine following transfection with the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter and the glial fibrillary acidic protein(GFAP) promoter are the effective tumor-specific promoter of gene expression and thus may be useful in targeted gene therapy of Malignant glioma.
Methods The hTERT promoter, the adenoviral early region1A (E1A),the GFAP promoter and the NIS gene were amplified by PCR or DNA replication. Detected the transcriptional activity of the hTERT and GFAP gene promoter. The GFAP promoter and hNIS gene segments,the hTERT promoter and E1A gene were ligated, respectively,and package the recombinant adenovirus Ad-Tp-E1A-Gp-NIS. The U87and U251glioma cells was tranfected by Ad-Tp-ElA-Gp-NIS, then the selective replication ability of the conditionally replicative adenovirus was evaluated by plaque forming assay, and proceeded western blots,125I uptake and exflux, clonogenecity following131I treatment.At last, on the basis of the cells-level experimental data, we had established U87tumor xenografts in nude mice and proceeded radioiodine therapy in U87tumors nude mice.
Results Genes had cloned into plasmids. The activity of GFAP promoter and hTERT promoter had reached to as much as about60%and43%of SV40promoter,respectively. The recombined adenovirus Ad-Tp-E1A-Gp-NIS was constructed successfully and tranfected the glioma cells. The western blots revealed bands of approximately43,70,120,49,110kDa, consistent with the β-acting, GFAP, hTERT,hNIS and hTPO proteins. The adenovirus selective-eplication experiment was proved the efficiency of Ad-Tp-E1A-Gp-NIS and the hTERT promoter could controled the eplication of Ad-Tp-E1A-Gp-NIS in the cell-lines. After transfection with the hNIS gene, the cells could intake125I-iodide. In clonogenic assay, transfected cells were killed, compared to control cells (transfected with Ad-CMV-EGFP) after incubated with131I. The U87tumor nude mice was evaluated. After treatment with131I and injection of Ad-Tp-E1A-Gp-NIS, the survival period of U87tumor loading nude mice were prolonged and the growth of tumor in mice was inhibited. Rude mice harboring xenografts after injection of Ad-Tp-E1A-Gp-NIS, could taken the99mTc.
Conclusions The experiments demonstrated that after transfected replication-selective adeno viruses Ad-Tp-E1A-Gp-NIS, the therapy of131I was achieved effectively in malignant glioma cell lines following induction of tumor-specific iodide uptake activity by GFAP promoters directed hNIS gene expression in vitro and in vivo.
引文
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