hTERT启动子调控hNIS基因介导肺癌细胞碘摄取和^131Ⅰ治疗的实验研究

Radioiodide uptake and therapy of lung cancer cells mediated by human sodium iodide symporter gene

目的构建含人端粒酶反转录酶（hTERT）核心启动子调控的人钠／碘同向转运体（hNIS）基因重组腺病毒，并靶向转染至肺癌A549细胞中特异性表达。探讨hTERT启动子调控的hNIS基因介导放射性碘治疗肿瘤的可能性。方法应用AdEasy系统构建重组腺病毒Ad-hTERT-hNIS，同时构建巨细胞病毒（CMV）启动子调控的hNIS重组腺病毒Ad-CMV-hNIS作为阳性对照，不含hNIS的重组腺病毒Ad-CMV作为阴性对照。应用反转录-聚合酶链反应（RT-PCR）方法验证hTERT在转染肿瘤细胞中的转录活性，摄碘实验检测表达的hNIS蛋白功能，细胞克隆形成实验评价^131Ⅰ对转染肿瘤细胞的毒性作用。结果成功构建重组腺病毒Ad-hTERT-hNIS、Ad-CMV-hNIS及Ad-CMV，并经PCR验证正确。RT-PCR证实hNIScDNA能从Ad·hTERT-hNIS转染的细胞中扩增出来。Ad-hTERT-hNIS和Ad-CMV-hNIS转染的肺癌A549细胞摄碘能力比阴性对照组Ad-CMV转染的细胞分别提高了23和31倍，且摄碘能力可以被NaClO4抑制。Ad-hTERT-hNIS和Ad-CMV-hNIS转染的肺癌A549细胞均可被^131Ⅰ杀死，2组细胞成活率分别为（31．2±1．45）％和（23．6±4．08）％，而阴性对照组和未转染病毒组分别为（89．0±2．99）％和（91．2±4．63）％。结论hTERT启动子调控的hNIS重组腺病毒转染肿瘤细胞后，应用^131Ⅰ治疗有望成为一种新的基因靶向治疗手段。

Objective Expression of the human sodium/iodide symporter （hNIS） in the thyroid gland provides for effective imaging and treatment of thyroid cancer using radiolabeled iodide. Transfer of hNIS gene into other tumors would expand the utility of this treatment to tumors of nonthyroid origin. The human telomerase reverse transcriptase （hTERT） is overexpressed in tumor cells, but is not expressed in normal cells. The aim of this study was to construct a recombinant adenovirus containing the hNIS under the control of hTERT core promoter to express specially on tumor cells, and to investigate the feasibility of radioiodine therapy mediated by hNIS gene controlled with hTERT promoter. Methods The recombinant adeno- virus Ad-hTERT-hNIS was constructed by AdEasy system. In addition, a positive control of adenovirus Adcytomegalovirus （CMV）-hNIS containing the CMV promoter followed with hNIS gene and a negative control of adenovirus Ad-CMV not containing hNIS gene were created by AdEasy system. The cell-specific transcriptional activity of hTERT was examined by reverse transcriptase-polymerase chain reaction （RT-PCR） in transiently transfected A549 cell lines. Iodide uptake assays were used to confirm hNIS expression and function. Toxic effects of ^131I on tumor cells were studied by in vitro clonogenic assay. Results Recombinant Ad-hTERT-hNIS was correctly constructed and confirmed by restriction enzyme analysis and PCR. RT-PCR showed that hNIS cDNA could be amplified by the transfected cells. A 23-fold and 31-fold increase in iodide uptake was observed in Ad-hTERT-hNIS and Ad-CMV-hNIS infected A549 cells, respectively. No significant iodide uptake increase was detected in cells infected with the negative control virus. The iodide uptake in transfected cells was inhibited by sodium perchlorate. In vitro clonogenic assay revealed that approximately 70% [ （31.2±1.45 ） % A549 cells alive ] of Ad-hTERT-hNIS and 80% [ （23.6±4.08） % A549 cells alive ] of Ad-CMV-hNIS transfected A549 cells were killed by exposure to ^131I, respectively, while only about 10% of NIS-negative control cells were killed [ （89.0±2.99）% A：549 cells alive for Ad-CMV group and （91.2±4. 63）% A549 cells alive for no-infected group ]. Conclusion Radioiodine therapy following tumor cells transfected with recombinant adenovirus Ad-hTERT-hNIS may likely be a successful entity benefited by this novel gene targeting therapy strategy.