携带人NIS基因和TPO基因的重组腺病毒联合转染肝癌细胞介导放射性碘摄取和有机化的基础研究
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摘要
目的:
NIS基因转染非甲状腺肿瘤介导放射性核素内放射治疗成为肿瘤基因治疗的又一研究方向。尽管转染了NIS基因的肿瘤细胞或病灶,均能迅速大量摄取放射性碘,但碘在细胞或病灶内停留时间短,疗效不理想。我们利用腺病毒载体将NIS和TPO基因联合转染肝癌细胞,试图延长放射性碘在转染细胞内的储留时间,达到提高疗效的目的。
方法:
1.用RT-PCR方法从Graves’甲亢病人甲状腺组织中扩增出hNIS和hTPO基因,亚克隆到质粒载体,构建成pGEM-Teasy-NIS重组质粒和PBK-CMV-TPO重组质粒,通过酶切和DNA序列分析进行鉴定。
2.将hNIS和hTPO基因分别克隆到腺病毒AdEasier系统的穿梭质粒pAdTrack-CMV上,通过在大肠杆菌BJ51 83内同骨架质粒pAdEasy-1同源重组、并在293细胞中包装、扩增,纯化、构建携带hNIS和hTPO的重组腺病毒AdNIS和AdTPO,测定病毒滴度。Western-Blotting检测重组腺病毒AdNIS和AdTPO的表达。
3.AdNIS单独转染,AdNIS和AdTPO联合转染肝癌细胞HepG2,确定最适MOI,动态检测~(125)I的摄取、观测NaClO_4对碘摄取抑制情况、TCA蛋白沉淀检测碘化蛋白、动态检测碘排泄情况,细胞克隆形成实验评价单独和联合转染~(131)I细胞毒性作用。
Objective:~(131)I therapy is a widely accepted treatment for differentiated thyroid cancer. Transfer of the NIS into nonthyroid tumors by coupling radioactive iodide administration has been proposed as a new principle of cancer gene therapy. Although efficient iodide uptake is achieved in the tumors, no obvious therapeutic effect could be observed with ~(131)I, most probably because the iodide retention time in the target cells is short. We propose to accumulation and organify the iodide by coexpression NIS and TPO in hepatocellular cancer cells to circumvent this problem. Mehtods:1. hNIS and hTPO coding region were amplified from Graves' patient's thyroid tissue by RT-PCR, subcloned into plasmid vector, then constructed recombinant plasmid pGEM-Teasy-NIS and PBK-CMV-TPO , and verify them by restriction enzyme analysis and DNA sequencing.2. hNIS and hTPO coding region were subcloned into shuttle plasmid pAdTrack-CMV which contained a green fluorescent protein (GFP) gene respectively, and further homologous recombined in the bacteria BJ5183 that already contained AdEasy-1 plasmid . Positive recombinant adenovirus vectors were selected, packaged, and amplified in the 293 cells. After purification, viral titers were calculated. Using Western-Blotting to test the protein expressing of AdNIS and AdTPO.
3. HepG2 cells were transfected with AdNIS or cotransfected with AdNIS and AdTPO. Iodide uptake and efflux were evaluated, cell killing with 131I and colongenic assay were also assessed between two groups.4. Intratumoral injection AdNIS in tumor-bearing nude mice, expression of the NIS protein in the tumor was confirmed by immunohistological analysis. Organs' radioactivity were counted after injected ' 5I, then calculated the percentage of injected dose per gram (%ID/g).' I was administered intravenously through the tail vein for the imaging of tumor-bearing nude mice.Results:1. Recombinant plasmids pGEM-Teasy-NIS and PBK-CMV-TPO were correctly constructed and confirmed by restriction enzyme analysis and DNA Sequencing2. Recombinant AdNIS and AdTPO were correctly constructed and confirmed by restriction enzyme analysis and PCR. The viral titer was 1.5 X 109 efu/ml, 2.0 X 109efu/ml respectively. Special 90kD and HOkD protein were found by SDS-PAGE electrophoresis, and could immunoreact with mouse monoclonal hNIS and hTPO-specific antibody, respectively.3. The cells transfected with AdNIS showed 125I uptake capability, 24 times higher than that of noninfected cells. Iodide uptake could be specifically inhibited by perchlorate anions.Radioiodide efflux was rapid ,with a T1/2 efflux of 16min, co-transfection with AdNIS and AdTPO genes could not increase iodide uptake (p > 0.05) , but could organify partial radioiodide, which rentention in the cell was mildly increase,Ti/2 efflUx ~25min, 40 %of AdNIS-transfected tumor cells were selectively killed by 131I, while greater cytotoxicity was observed in cotransfected tumor cells.4. Immunohistological analysis confirmed the expression of the NIS protein in the tumor and localization in the surface of cells. AdNIS-treated tumor uptake of 125I was shown (10.10 ± 1.81%ID/g) at 2 h after injection, the biological half-life was 2.35h. Imaging study of tumor-bearing nude mice model showed tumor could be clearly observed with 13II at 2h after injection.
Conclusion:hNIS and hTPO coding region were successfully cloned and adenovirus AdNIS and AdTPO were constructed by using convenient Adeasier system. The cells transfected with AdNIS showed 125I uptake capability, cotransfection with AdNIS and AdTPO could organify partial radioiodide, but could not significantly improve radioiodide rentenion in the targeted cells. Tumor could uptake radioiodide and clear tumor imaging was performed. However, the retention time was short, which suggests should choose other proper shorter physical half-life radionuclide to obtain efficient treatment.
NIS基因转染非甲状腺肿瘤介导放射性核素内放射治疗成为肿瘤基因治疗的又一研究方向。尽管转染了NIS基因的肿瘤细胞或病灶,均能迅速大量摄取放射性碘,但碘在细胞或病灶内停留时间短,疗效不理想。我们利用腺病毒载体将NIS和TPO基因联合转染肝癌细胞,试图延长放射性碘在转染细胞内的储留时间,达到提高疗效的目的。
方法:
1.用RT-PCR方法从Graves’甲亢病人甲状腺组织中扩增出hNIS和hTPO基因,亚克隆到质粒载体,构建成pGEM-Teasy-NIS重组质粒和PBK-CMV-TPO重组质粒,通过酶切和DNA序列分析进行鉴定。
2.将hNIS和hTPO基因分别克隆到腺病毒AdEasier系统的穿梭质粒pAdTrack-CMV上,通过在大肠杆菌BJ51 83内同骨架质粒pAdEasy-1同源重组、并在293细胞中包装、扩增,纯化、构建携带hNIS和hTPO的重组腺病毒AdNIS和AdTPO,测定病毒滴度。Western-Blotting检测重组腺病毒AdNIS和AdTPO的表达。
3.AdNIS单独转染,AdNIS和AdTPO联合转染肝癌细胞HepG2,确定最适MOI,动态检测~(125)I的摄取、观测NaClO_4对碘摄取抑制情况、TCA蛋白沉淀检测碘化蛋白、动态检测碘排泄情况,细胞克隆形成实验评价单独和联合转染~(131)I细胞毒性作用。
Objective:~(131)I therapy is a widely accepted treatment for differentiated thyroid cancer. Transfer of the NIS into nonthyroid tumors by coupling radioactive iodide administration has been proposed as a new principle of cancer gene therapy. Although efficient iodide uptake is achieved in the tumors, no obvious therapeutic effect could be observed with ~(131)I, most probably because the iodide retention time in the target cells is short. We propose to accumulation and organify the iodide by coexpression NIS and TPO in hepatocellular cancer cells to circumvent this problem. Mehtods:1. hNIS and hTPO coding region were amplified from Graves' patient's thyroid tissue by RT-PCR, subcloned into plasmid vector, then constructed recombinant plasmid pGEM-Teasy-NIS and PBK-CMV-TPO , and verify them by restriction enzyme analysis and DNA sequencing.2. hNIS and hTPO coding region were subcloned into shuttle plasmid pAdTrack-CMV which contained a green fluorescent protein (GFP) gene respectively, and further homologous recombined in the bacteria BJ5183 that already contained AdEasy-1 plasmid . Positive recombinant adenovirus vectors were selected, packaged, and amplified in the 293 cells. After purification, viral titers were calculated. Using Western-Blotting to test the protein expressing of AdNIS and AdTPO.
3. HepG2 cells were transfected with AdNIS or cotransfected with AdNIS and AdTPO. Iodide uptake and efflux were evaluated, cell killing with 131I and colongenic assay were also assessed between two groups.4. Intratumoral injection AdNIS in tumor-bearing nude mice, expression of the NIS protein in the tumor was confirmed by immunohistological analysis. Organs' radioactivity were counted after injected ' 5I, then calculated the percentage of injected dose per gram (%ID/g).' I was administered intravenously through the tail vein for the imaging of tumor-bearing nude mice.Results:1. Recombinant plasmids pGEM-Teasy-NIS and PBK-CMV-TPO were correctly constructed and confirmed by restriction enzyme analysis and DNA Sequencing2. Recombinant AdNIS and AdTPO were correctly constructed and confirmed by restriction enzyme analysis and PCR. The viral titer was 1.5 X 109 efu/ml, 2.0 X 109efu/ml respectively. Special 90kD and HOkD protein were found by SDS-PAGE electrophoresis, and could immunoreact with mouse monoclonal hNIS and hTPO-specific antibody, respectively.3. The cells transfected with AdNIS showed 125I uptake capability, 24 times higher than that of noninfected cells. Iodide uptake could be specifically inhibited by perchlorate anions.Radioiodide efflux was rapid ,with a T1/2 efflux of 16min, co-transfection with AdNIS and AdTPO genes could not increase iodide uptake (p > 0.05) , but could organify partial radioiodide, which rentention in the cell was mildly increase,Ti/2 efflUx ~25min, 40 %of AdNIS-transfected tumor cells were selectively killed by 131I, while greater cytotoxicity was observed in cotransfected tumor cells.4. Immunohistological analysis confirmed the expression of the NIS protein in the tumor and localization in the surface of cells. AdNIS-treated tumor uptake of 125I was shown (10.10 ± 1.81%ID/g) at 2 h after injection, the biological half-life was 2.35h. Imaging study of tumor-bearing nude mice model showed tumor could be clearly observed with 13II at 2h after injection.
Conclusion:hNIS and hTPO coding region were successfully cloned and adenovirus AdNIS and AdTPO were constructed by using convenient Adeasier system. The cells transfected with AdNIS showed 125I uptake capability, cotransfection with AdNIS and AdTPO could organify partial radioiodide, but could not significantly improve radioiodide rentenion in the targeted cells. Tumor could uptake radioiodide and clear tumor imaging was performed. However, the retention time was short, which suggests should choose other proper shorter physical half-life radionuclide to obtain efficient treatment.
引文
1. Larson SM, Krenning EP. A pragmatic perspective on molecular targeted radionuclide therapy. J Nucl Med 2005; 46 Suppl 1:1S-3S
2. Buchsbaum DJ, Chaudhuri TR, Zinn KR. Radiotargeted gene therapy. J Nucl Med 2005; 46 Suppl 1:179S-86S.
3. Mairs RJ, Boyd M. Targeting Radiotherapy to Cancer by Gene Transfer. J Biomed Biotechnol 2003; 2003(2):102-9.
4. Boyd M, Spenning HS, Mairs RJ. Radiation and gene therapy: rays of hope for the new millennium? Curr Gene Ther 2003; 3(4):319-39.
5.匡安仁,谭天秩。放射性核素治疗的现状和思考。中华核医学杂志,2003;23 (6):325～326。
6. Carrasco N. Iodide transport in the thyroid gland. Biochim Biophys Acta 1993; 1154(1):65-82.
7. Smanik PA, Liu Q, Furminger TL, et al. Cloning of the human sodium lodide symporter. Biochem Biophys Res Commun 1996; 226(2):339-45.
8. Dai G, Levy O, Carrasco N. Cloning and characterization of the thyroid iodide transporter. Nature 1996; 379(6564):458-60.
9. Eskandari S, Loo DD, Dai G, et al. Thyroid Na+/I-symporter. Mechanism, stoichiometry, and specificity. J Biol Chem 1997; 272(43):27230-8.
10. Kogai T, Endo T, Saito T, Miyazaki A, Kawaguchi A, Onaya T. Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells. Endocrinology 1997; 138(6):2227-32.
11. Suzuki K, Mori A, Lavaroni S. Thyroglobulin regulates follicular function and
??heterogeneity by suppressing thyroid-specific gene expression. Biochimie 1999;81(4):329-40.
12. Pekary AE, Hershman JM. Tumor necrosis factor, ceramide, transforming growth factor-betal, and aging reduce Na+/I- symporter messenger ribonucleic acid levels in FRTL-5 cells. Endocrinology 1998;139(2):703-12.
13. Schmutzler C, Winzer R, Meissner-Weigl J, Kohrle J. Retinoic acid increases sodium/iodide symporter mRNA levels in human thyroid cancer cell lines and suppresses expression of functional symporter in nontransformed FRTL-5 rat thyroid cells. Biochem Biophys Res Commun 1997;240(3):832-8.
14. Mandell RB, Mandell LZ, Link Ci, Jr. Radioisotope concentrator gene therapy using the sodium/iodide symporter gene. Cancer Res 1999;59(3):661-8.
15. Nakamoto Y, Saga T, Misaki T, et al. Establishment and characterization of a breast cancer cell line expressing Na+/I- symporters for radioiodide concentrator gene therapy. J Nucl Med 2000;41(11):1898-904.
16. Boland A, Ricard M, Opolon P, et al. Adenovirus-mediated transfer of the thyroid sodium/iodide symporter gene into tumors for a targeted radiotherapy. Cancer Res 2000;60(13):3484-92.
17. Shimura H, Haraguchi K, Miyazaki A, Endo T, Onaya T. Iodide uptake and experimental 131I therapy in transplanted undifferentiated thyroid cancer cells expressing the Na+/I-symporter gene. Endocrinology 1997;138(10):4493-6.
18. Maxon HR, Thomas SR, Hertzberg VS, et al. Relation between effective radiation dose and outcome of radioiodine therapy for thyroid cancer. N Engl J Med 1983;309(16):937-41.
19. Berman M, Hoff E, Barandes M, et al. Iodine kinetics in man~a model. J Clin Endocrinol Metab 1968;28(1):1-14.
20. Xing Y, Kuang A. Development of studies of TPO gene and its application in nuclear medicine. Nucl Med Commun 2003;24(8):853-6.
21. El-Aneed A. Current strategies in cancer gene therapy. Eur J Pharmacol 2004;498(1-3):1-8.
22. Rosenberg SA, Aebersold P, Cornetta K, et al. Gene transfer into humans-immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med 1990;323(9):570-8.
23. Parney IF, Chang LJ. Cancer immunogene therapy: a review. J Biomed Sci 2003;10(1):37-43.
24. Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene. 2003;22(56):9087-96.
25. Kobayashi H, Takemura Y, Miyachi H. Novel approaches to reversing anti-cancer drug resistance using gene-specific therapeutics. Hum Cell 2001; 14(3):172-84.
26. Yazawa K, Fisher WE, Brunicardi FC. Current progress in suicide gene therapy for cancer. World J Surg 2002; 26(7):783-9.
27. Scappaticci FA. The therapeutic potential of novel antiangiogenic therapies. Expert Opin Investig Drugs 2003; 12(6):923-32.
28. Spitzweg C, Joba W, Eisenmenger W, et al. Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa. J Clin Endocrinol Metab 1998; 83(5): 1746-51.
29. Magnusson RP, Chazenbalk GD, Gestautas J, et al. Molecular cloning of the complementary deoxyribonucleic acid for human thyroid peroxidase. Mol Endocrinol 1987; 1(11):856-61.
30. Ferrand M, Le Fourn V, Franc JL. Increasing diversity of human thyroperoxidase generated by alternative splicing. Characterized by molecular cloning of new transcripts with singleand multispliced mRNAs. J Biol Chem 2003; 278(6):3793-800.
31. Gerard CM, Lefort A, Christophe D, et al. Distinct transcriptional effects of cAMP on 2 thyroid specific genes: thyroperoxidase and thyroglobulin. Horm Metab Res Suppl 1990; 23:38-43.
32. Napolitano G, Montani V, Giuliani C, et al. Transforming growth factor-betal down-regulation of major histocompatibility complex class I in thyrocytes: coordinate regulation of two separate elements by thyroid-specific as well as ubiquitous transcription factors. Mol Endocrinol 2000; 14(4):486-505.
33. Miyazaki A, Shimura H, Endo T, Haraguchi K, Onaya T. Tumor necrosis factor-alpha and interferon-gamma suppress both gene expression and deoxyribonucleic acid-binding of TTF-2 in FRTL-5 cells. Endocrinology 1999; 140(9):4214-20.
34. Ortiz L, Zannini M, Di Lauro R, Santisteban P. Transcriptional control of the forkhead thyroid transcription factor TTF-2 by thyrotropin, insulin, and insulin-like growth factor Ⅰ. J Biol Chem 1997; 272(37):23334-9.
35. Lazar V, Bidart JM, Caillou B, et al. Expression of the Na+/I- symporter gene in human thyroid tumors: a comparison study with other thyroid-specific genes. J Clin Endocrinol Metab 1999; 84(9):3228-34.
36.Carl W. Dieffenbach, Gabriela S. Dveksler.降落PCR.PCR技术实验指南[M].第2版.美国冷泉港实验室出版社,2003;34。
37. Zhang WW. Development and application of adenoviral vectors for gene therapy of cancer. Cancer Gene Ther 1999; 6(2): 113-38.
38. St George JA. Gene therapy progress and prospects: adenoviral vectors. Gene Ther 2003; 10(14):1135-41.
39. He TC, Zhou S, da Costa LT, Yu J, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 1998; 95(5):2509-14.
40. Harrington KJ, Bateman AR, Melcher AA, et al. Cancer gene therapy: Part 1. Vector development and regulation of gene expression. Clin Oncol (R Coll Radiol) 2002; 14(1):3-16.
41. Tomanin R, Scarpa M. Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction. Curt Gene Ther 2004; 4(4):357-72.
42. Graham FL, Prevec L. Methods for construction of adenovirus vectors. Mol Biotechnol 1995; 3(3):207-20.
43. Bett AJ, Haddara W, Prevec L, et al. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A 1994; 91(19):8802-6.
44. Chung JK. Sodium iodide symporter: its role in nuclear medicine. J Nucl Med 2002; 43(9): 1188-200.
45. Dadachova E, Carrasco N. The Na/I symporter (NIS): imaging and therapeutic applications, Semin Nucl Med 2004; 34(1):23-31.
46. Haberkorn U, Kinscherf R, Kissel M, et al. Enhanced iodide transport after transfer of the human sodium iodide symporter gene is associated with lack of retention and low absorbed dose. Gene Ther 2003; 10(9):774-80.
47.吴孟超。原发性肝癌外科综合治疗的现状和展望。中华外科杂志 2004;42(1):13-15。
48.林芷英。原发性肝Ca的化疗及研究进展。中国医院用药评价与分析 2004;4 (1):50-52。
49. Weiss SJ, Philp NJ, Grollman EF. Iodide transport in a continuous line of cultured cells from rat thyroid. Endocrinology 1984; 114(4): 1090-8.
50.朱玲锦,管昌田。甲状腺功能亢进症。中医古籍出版社,2003;13-14。
51. Schipper ML, Weber A, Behe M, et al. Radioiodide treatment after sodium iodide symporter gene transfer is a highly effective therapy in neuroendocrine tumor ceils. Cancer Res 2003; 63(6): 1333-8.
52. Boland A, Magnon C, Filetti S, et al. Transposition of the thyroid iodide uptake and organification system in nonthyroid tumor cells by adenoviral vector-mediated gene transfers. Thyroid 2002; 12(1): 19-26.
53. Yeang HY, Yusof F, Abdullah L. Precipitation of Hevea brasiliensis latex proteins with trichloroacetic acid and phosphotungstic acid in preparation for the Lowry protein assay. Anal Biochem 1995; 226(1):35-43.
54. Huang M, Batra R K, Kogai T, et al. Ectopic expression of the thyroperoxidase gene augments radioiodide uptake and retention mediated by the sodium iodide symporter in non-small cell lung cancer. Cancer Gene Ther 2001; 8(8):612-618.
55. Dadachova E, Carrasco N. The Na/I symporter (NIS): imaging and therapeutic applications. Semin Nucl Med 2004; 34(1):23-31.
56. Dingli D, Russell SJ, Morris JC, 3rd. In vivo imaging and tumor therapy with the sodium iodide symporter. J Cell Biochem 2003; 90(6):1079-86.
57. Haberkorn U, Beuter P, Kubler W, et al. Iodide kinetics and dosimetry in vivo after transfer of the human sodium iodide symporter gene in rat thyroid carcinoma cells. J Nucl Med 2004; 45(5):827-33.
58.杨吉成,谬竞试,阮长耿。第十三章:基因治疗。化学工业出版社。2002:190-210。
59. Smit JW, Schroder-van der Elst JP, Karperien M, et al. Iodide kinetics and experimental (131)I therapy in a xenotransplanted human sodium-iodide symporter-transfected human follicular thyroid carcinoma cell line. J Clin Endocrinol Metab 2002; 87(3): 1247-53.
60. Spitzweg C, Dietz AB, O'Connor M K, et al. In vivo sodium iodide symporter gene therapy of prostate cancer. Gene Ther 2001; 8(20): 1524-1531.
61. Dingli D, Peng KW, Harvey ME, et al. Image-guided radiovirotherapy for multiple myeloma using a recombinant measles virus expressing the thyroidal sodium iodide symporter. Blood 2004; 103(5): 1641-6.
62. Dadachova E, Bouzahzah B, Zuckier L S, et al. Rhenium-188 as an alternative to Iodine-131 for treatment of breast tumors expressing the sodium/iodide symporter (NIS). Nucl Med Biol 2002; 29(1):13-18.
63. Carlin S, Mairs R J, Welsh P, et al. Sodium-iodide symporter (NIS)-mediated accumulation of [211At]astatide in NIS-transfected human cancer cells. Nucl Med Biol 2002; 29(7):729-739.
64. Carlin S, Akabani G, Zalutsky MR. In vitro cytotoxicity of (211)At-astatide and (131)I-iodide to glioma tumor cells expressing the sodium/iodide symporter. J Nucl Med 2003; 44(11): 1827-38.
2. Buchsbaum DJ, Chaudhuri TR, Zinn KR. Radiotargeted gene therapy. J Nucl Med 2005; 46 Suppl 1:179S-86S.
3. Mairs RJ, Boyd M. Targeting Radiotherapy to Cancer by Gene Transfer. J Biomed Biotechnol 2003; 2003(2):102-9.
4. Boyd M, Spenning HS, Mairs RJ. Radiation and gene therapy: rays of hope for the new millennium? Curr Gene Ther 2003; 3(4):319-39.
5.匡安仁,谭天秩。放射性核素治疗的现状和思考。中华核医学杂志,2003;23 (6):325～326。
6. Carrasco N. Iodide transport in the thyroid gland. Biochim Biophys Acta 1993; 1154(1):65-82.
7. Smanik PA, Liu Q, Furminger TL, et al. Cloning of the human sodium lodide symporter. Biochem Biophys Res Commun 1996; 226(2):339-45.
8. Dai G, Levy O, Carrasco N. Cloning and characterization of the thyroid iodide transporter. Nature 1996; 379(6564):458-60.
9. Eskandari S, Loo DD, Dai G, et al. Thyroid Na+/I-symporter. Mechanism, stoichiometry, and specificity. J Biol Chem 1997; 272(43):27230-8.
10. Kogai T, Endo T, Saito T, Miyazaki A, Kawaguchi A, Onaya T. Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells. Endocrinology 1997; 138(6):2227-32.
11. Suzuki K, Mori A, Lavaroni S. Thyroglobulin regulates follicular function and
??heterogeneity by suppressing thyroid-specific gene expression. Biochimie 1999;81(4):329-40.
12. Pekary AE, Hershman JM. Tumor necrosis factor, ceramide, transforming growth factor-betal, and aging reduce Na+/I- symporter messenger ribonucleic acid levels in FRTL-5 cells. Endocrinology 1998;139(2):703-12.
13. Schmutzler C, Winzer R, Meissner-Weigl J, Kohrle J. Retinoic acid increases sodium/iodide symporter mRNA levels in human thyroid cancer cell lines and suppresses expression of functional symporter in nontransformed FRTL-5 rat thyroid cells. Biochem Biophys Res Commun 1997;240(3):832-8.
14. Mandell RB, Mandell LZ, Link Ci, Jr. Radioisotope concentrator gene therapy using the sodium/iodide symporter gene. Cancer Res 1999;59(3):661-8.
15. Nakamoto Y, Saga T, Misaki T, et al. Establishment and characterization of a breast cancer cell line expressing Na+/I- symporters for radioiodide concentrator gene therapy. J Nucl Med 2000;41(11):1898-904.
16. Boland A, Ricard M, Opolon P, et al. Adenovirus-mediated transfer of the thyroid sodium/iodide symporter gene into tumors for a targeted radiotherapy. Cancer Res 2000;60(13):3484-92.
17. Shimura H, Haraguchi K, Miyazaki A, Endo T, Onaya T. Iodide uptake and experimental 131I therapy in transplanted undifferentiated thyroid cancer cells expressing the Na+/I-symporter gene. Endocrinology 1997;138(10):4493-6.
18. Maxon HR, Thomas SR, Hertzberg VS, et al. Relation between effective radiation dose and outcome of radioiodine therapy for thyroid cancer. N Engl J Med 1983;309(16):937-41.
19. Berman M, Hoff E, Barandes M, et al. Iodine kinetics in man~a model. J Clin Endocrinol Metab 1968;28(1):1-14.
20. Xing Y, Kuang A. Development of studies of TPO gene and its application in nuclear medicine. Nucl Med Commun 2003;24(8):853-6.
21. El-Aneed A. Current strategies in cancer gene therapy. Eur J Pharmacol 2004;498(1-3):1-8.
22. Rosenberg SA, Aebersold P, Cornetta K, et al. Gene transfer into humans-immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med 1990;323(9):570-8.
23. Parney IF, Chang LJ. Cancer immunogene therapy: a review. J Biomed Sci 2003;10(1):37-43.
24. Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene. 2003;22(56):9087-96.
25. Kobayashi H, Takemura Y, Miyachi H. Novel approaches to reversing anti-cancer drug resistance using gene-specific therapeutics. Hum Cell 2001; 14(3):172-84.
26. Yazawa K, Fisher WE, Brunicardi FC. Current progress in suicide gene therapy for cancer. World J Surg 2002; 26(7):783-9.
27. Scappaticci FA. The therapeutic potential of novel antiangiogenic therapies. Expert Opin Investig Drugs 2003; 12(6):923-32.
28. Spitzweg C, Joba W, Eisenmenger W, et al. Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa. J Clin Endocrinol Metab 1998; 83(5): 1746-51.
29. Magnusson RP, Chazenbalk GD, Gestautas J, et al. Molecular cloning of the complementary deoxyribonucleic acid for human thyroid peroxidase. Mol Endocrinol 1987; 1(11):856-61.
30. Ferrand M, Le Fourn V, Franc JL. Increasing diversity of human thyroperoxidase generated by alternative splicing. Characterized by molecular cloning of new transcripts with singleand multispliced mRNAs. J Biol Chem 2003; 278(6):3793-800.
31. Gerard CM, Lefort A, Christophe D, et al. Distinct transcriptional effects of cAMP on 2 thyroid specific genes: thyroperoxidase and thyroglobulin. Horm Metab Res Suppl 1990; 23:38-43.
32. Napolitano G, Montani V, Giuliani C, et al. Transforming growth factor-betal down-regulation of major histocompatibility complex class I in thyrocytes: coordinate regulation of two separate elements by thyroid-specific as well as ubiquitous transcription factors. Mol Endocrinol 2000; 14(4):486-505.
33. Miyazaki A, Shimura H, Endo T, Haraguchi K, Onaya T. Tumor necrosis factor-alpha and interferon-gamma suppress both gene expression and deoxyribonucleic acid-binding of TTF-2 in FRTL-5 cells. Endocrinology 1999; 140(9):4214-20.
34. Ortiz L, Zannini M, Di Lauro R, Santisteban P. Transcriptional control of the forkhead thyroid transcription factor TTF-2 by thyrotropin, insulin, and insulin-like growth factor Ⅰ. J Biol Chem 1997; 272(37):23334-9.
35. Lazar V, Bidart JM, Caillou B, et al. Expression of the Na+/I- symporter gene in human thyroid tumors: a comparison study with other thyroid-specific genes. J Clin Endocrinol Metab 1999; 84(9):3228-34.
36.Carl W. Dieffenbach, Gabriela S. Dveksler.降落PCR.PCR技术实验指南[M].第2版.美国冷泉港实验室出版社,2003;34。
37. Zhang WW. Development and application of adenoviral vectors for gene therapy of cancer. Cancer Gene Ther 1999; 6(2): 113-38.
38. St George JA. Gene therapy progress and prospects: adenoviral vectors. Gene Ther 2003; 10(14):1135-41.
39. He TC, Zhou S, da Costa LT, Yu J, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 1998; 95(5):2509-14.
40. Harrington KJ, Bateman AR, Melcher AA, et al. Cancer gene therapy: Part 1. Vector development and regulation of gene expression. Clin Oncol (R Coll Radiol) 2002; 14(1):3-16.
41. Tomanin R, Scarpa M. Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction. Curt Gene Ther 2004; 4(4):357-72.
42. Graham FL, Prevec L. Methods for construction of adenovirus vectors. Mol Biotechnol 1995; 3(3):207-20.
43. Bett AJ, Haddara W, Prevec L, et al. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A 1994; 91(19):8802-6.
44. Chung JK. Sodium iodide symporter: its role in nuclear medicine. J Nucl Med 2002; 43(9): 1188-200.
45. Dadachova E, Carrasco N. The Na/I symporter (NIS): imaging and therapeutic applications, Semin Nucl Med 2004; 34(1):23-31.
46. Haberkorn U, Kinscherf R, Kissel M, et al. Enhanced iodide transport after transfer of the human sodium iodide symporter gene is associated with lack of retention and low absorbed dose. Gene Ther 2003; 10(9):774-80.
47.吴孟超。原发性肝癌外科综合治疗的现状和展望。中华外科杂志 2004;42(1):13-15。
48.林芷英。原发性肝Ca的化疗及研究进展。中国医院用药评价与分析 2004;4 (1):50-52。
49. Weiss SJ, Philp NJ, Grollman EF. Iodide transport in a continuous line of cultured cells from rat thyroid. Endocrinology 1984; 114(4): 1090-8.
50.朱玲锦,管昌田。甲状腺功能亢进症。中医古籍出版社,2003;13-14。
51. Schipper ML, Weber A, Behe M, et al. Radioiodide treatment after sodium iodide symporter gene transfer is a highly effective therapy in neuroendocrine tumor ceils. Cancer Res 2003; 63(6): 1333-8.
52. Boland A, Magnon C, Filetti S, et al. Transposition of the thyroid iodide uptake and organification system in nonthyroid tumor cells by adenoviral vector-mediated gene transfers. Thyroid 2002; 12(1): 19-26.
53. Yeang HY, Yusof F, Abdullah L. Precipitation of Hevea brasiliensis latex proteins with trichloroacetic acid and phosphotungstic acid in preparation for the Lowry protein assay. Anal Biochem 1995; 226(1):35-43.
54. Huang M, Batra R K, Kogai T, et al. Ectopic expression of the thyroperoxidase gene augments radioiodide uptake and retention mediated by the sodium iodide symporter in non-small cell lung cancer. Cancer Gene Ther 2001; 8(8):612-618.
55. Dadachova E, Carrasco N. The Na/I symporter (NIS): imaging and therapeutic applications. Semin Nucl Med 2004; 34(1):23-31.
56. Dingli D, Russell SJ, Morris JC, 3rd. In vivo imaging and tumor therapy with the sodium iodide symporter. J Cell Biochem 2003; 90(6):1079-86.
57. Haberkorn U, Beuter P, Kubler W, et al. Iodide kinetics and dosimetry in vivo after transfer of the human sodium iodide symporter gene in rat thyroid carcinoma cells. J Nucl Med 2004; 45(5):827-33.
58.杨吉成,谬竞试,阮长耿。第十三章:基因治疗。化学工业出版社。2002:190-210。
59. Smit JW, Schroder-van der Elst JP, Karperien M, et al. Iodide kinetics and experimental (131)I therapy in a xenotransplanted human sodium-iodide symporter-transfected human follicular thyroid carcinoma cell line. J Clin Endocrinol Metab 2002; 87(3): 1247-53.
60. Spitzweg C, Dietz AB, O'Connor M K, et al. In vivo sodium iodide symporter gene therapy of prostate cancer. Gene Ther 2001; 8(20): 1524-1531.
61. Dingli D, Peng KW, Harvey ME, et al. Image-guided radiovirotherapy for multiple myeloma using a recombinant measles virus expressing the thyroidal sodium iodide symporter. Blood 2004; 103(5): 1641-6.
62. Dadachova E, Bouzahzah B, Zuckier L S, et al. Rhenium-188 as an alternative to Iodine-131 for treatment of breast tumors expressing the sodium/iodide symporter (NIS). Nucl Med Biol 2002; 29(1):13-18.
63. Carlin S, Mairs R J, Welsh P, et al. Sodium-iodide symporter (NIS)-mediated accumulation of [211At]astatide in NIS-transfected human cancer cells. Nucl Med Biol 2002; 29(7):729-739.
64. Carlin S, Akabani G, Zalutsky MR. In vitro cytotoxicity of (211)At-astatide and (131)I-iodide to glioma tumor cells expressing the sodium/iodide symporter. J Nucl Med 2003; 44(11): 1827-38.