多聚乙烯基亚胺介导的自杀基因系统对卵巢癌靶向杀伤效应的研究
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摘要
随着肿瘤基因治疗研究的迅速发展,卵巢癌的基因治疗也成为近年来的研究热点。目前肿瘤基因治疗领域存在的关键技术问题是如何提高基因转导系统的高效性和靶向性。多聚乙烯基亚胺(polyethylenimine, PEI)作为近年来一种新型的非病毒载体,具有较高的转导效率和较低的细胞毒性,为基因转移提供了一条有效的新途径。以肿瘤特异性启动子hTERT和卵巢组织特异性启动子OSP-1调控下的自杀基因系统是卵巢癌基因治疗中的靶向性策略之一。其次在肿瘤的基因研究中,建立动物模型来研究肿瘤的发生、发展以及各种治疗手段的效果,是临床前肿瘤研究的必经之路。而在现有的肿瘤研究中,与临床相关的动物模型却存在着诸多问题。近年来生物荧光成像系统(bioluminescent imaging, BLI)作为一种新开发的动物模型,具有快速、灵敏、无创和高分辨率的特点,为肿瘤研究提供了良好的技术平台。本课题以多聚乙烯基亚胺为载体,介导靶向性的自杀基因系统转导人卵巢癌细胞,并利用生物荧光成像系统建立动物模型,研究其转导效率和靶向抗瘤效应。全文共分为两部分,现分部叙述如下。
第一部分 多聚乙烯基亚胺介导的卵巢特异性启动子调控下的自杀基因抑制卵巢癌生长的实验研究
目的:应用多聚乙烯基亚胺将卵巢特异性启动子OSP-1调控下的自杀基因HSV-tk导入人卵巢癌细胞,探讨其体外、体内的抗瘤效应和靶向性。
方法:(1)将pGL3-Luc质粒分别介导多聚乙烯基亚胺PEI、脂质体DOTAP和裸DNA转染人卵巢癌细胞SKOV3,检测荧光素酶表达RLU(relative luciferase unit);(2)利用PEI将卵巢特异性启动子调控下的真核表达质粒pOSP1-HSVtk
Recently with the rapid development of tumor gene therapy, the reseach on ovarian cancer has also become a hotspot. To improve the transfection efficiency and targeted delivery of gene vector have become two critical problems in the field of gene therapy. Many approaches have been developed to solve these problems. Polycationic polyethylenimine (PEI), as a new kind of nonviral gene vector, has provided an effective solution with its higher transfection efficiency and lower toxicity. The suicide gene therapy with a tumor specific promoter (hTERT) or an ovarian specific promoter (OSP1) is one of these targeted delivery strategies for ovarian cancer. In addition, establishing some relevant animal models is a necessary method in preclinical oncology research in order to observate tumor growth and effect before or after some treatment in the field of tumor gene therapy. However, the relevant animal models remain many question and challenge at present. Fortunately, bioluminescent imaging (BLI), as a new kind of animal model with its sensitive, rapid, less invasive and greater accurate, has provided a excellent technical solution. This subject aimed to investigate the transfection efficiency and targeted killing activity using specific suicide gene therapy system and polyethylenimine mediated transfection by establishing animal model of BLI for ovarian cancer. This subject was divided into two parts and the relevant abstract was as follows.
PART ⅠANTI-TUMOR STUDY OF SUICIDE GENE THERAPY WITH AN OVARIAN-SPECIFIC PROMOTER AND POLYETHYLENIMINE MEDIATED TRANSFECTION FOR OVARIAN CANCEROBJECTIVE: To investigate the anti-tumor and targeted delivery effects of HSV-tk suicide gene therapy adopting an ovarian-specific promoter (OSPl) for human ovarian carcinoma by polyethylenimine-mediated transfection in vitro and in vivo.METHODS: (1) Human ovarian carcinoma SKOV3 cells were transfected by pGL3-Luc plasmid mediated with polyethylenimine (PEI), DOTAP liposome and naked DNA respectively. The transfection efficiency was measured by RLU (relative luciferase unit); (2) The pOSPl-HSVtk plasmid containing an ovarian-specific promoter was transfected into the human ovarian carcinoma cell SKOV3 by PEI, human lung carcinoma cell NCI-H460 and human heptocellular carcinoma cell HepG2 and SMMC7721. The cytotoxicities resulted from GCV were evaluated using the MTT assay. The concentrations of GCV in SKOV3 cells were detected by high performance liquid chromatography (HPLC). The apoptosis of SKOV3 cells were observed by flow cytometry (FCM) and TdT-mediated dUTP nick end labeling (TUNEL) technique; (3) SKOV3 ovarian cancer xenograft model was established in BALB/C nude mice. The tumors were transfected with pOSPl-HSVtk using PEI as the transfection agent. The expressed TK activities were estimated by monitoring the GCV concentration change using a HPLC assay; (4) The weight of the nude mice, the tumor volumes and tumor weights were all recorded to calculate the tumor inhibition rates by weight and by volume. Histopathological analysis and TUNEL method were also performed; (5) SKOV3 cells were implanted intraperitoneally to BALB/C nude mice, then the survival period and survival prolong rate of BALB/C nude mice were evaluated the efficacy of PEI mediated pOSP1-HSVtk gene delivery followed by GCV treatment in vivo.RESULTS: (1)The polyethylenimine group had the highest luciferase activity
(187.35 ± 6.48) compared with other two groups (45.74±5.98, 0.03 ± 0.00) with significant statistical difference(P<0.01); (2) GCV was shown to be toxicity only in SKOV3 cells, not in NCI-H460 cells, SMMC7721and HepG2 cells. The concentrations of GCV in SKOV3 cells detected by HPLC gradually decreased with increase of time. FCM showed the apoptotic rate of cells significantly increased in SKOV3 exposed to GCV 24h, 48h and 72h (8.42 ±0.76%, 18.50±1.78%, 34.80+ 3.46%). The substantial cell apoptosis in SKOV3 was confirmed by TUNEL; (3) Compared with the control group in vivo, GCV concentration in tumor tissues transfected pOSP1-HSVtk were shown to be significantly lower by HPLC (0.05>P>0.01); (4) The tumor volume and the tumor weight in the treated group were also significantly decreased (P<0.01). The tumor volume inhibition rate and the tumor weight inhibition rate were estimated to be 63.66% and 58.98% respectively. Histological examination revealed heavy haemorrhage and necrosis in the tumor tissues, and TUNEL confirmed substantial cell apoptosis in the treated group; (5) Compared with these control groups, the survival period of the treated group was remarkably longer in the ovarian cancer survival model (P<0.01). The survival prolong rate of treated group was 63.80%.CONCLUSION: The suicide gene therapy system using an ovarian-specific promoter by polyethylenimine mediated transfection had a tissue-specific killing activity for human ovarian cancer in vitro and in vivo, which indicated its potential to improve the efficiency and the targeting in the field of gene therapy.
PART ⅡANTI-TUMOR EFFICACY STUDY OF DOUBLE SUICIDE GENETHERAPY WITH A hTERT PROMOTER FOR OVARIANCANCER BY POLYETHYLENIMINE MEDIATEDTRANSFECTIONOBJECTIVE: To evaluate the killing activity of double suicide gene therapy adopting a human telomerase reverse transcriptase (hTERT) promoter and polyethylenimine mediated transfection for human ovarian carcinoma in vivo by establishing traditional SKOV3 intraperitoneal implantation animal model as well as SKOV3-Luciferase intraperitoneal animal mode of bioluminescent imaging (BLI) respectively.METHODS: (1) SKOV3 cells were implanted intraperitoneally to BALB/C nude mice and the ovarian cancer survival model was established. Diverse dose (5 μ g, 30 μ g, 50 μ g) of pBTdel-279-CD-TK were given by intraperitoneal injection using PEI as the transfection agent. Diverse treatments were started by intraperitoneal injection PEI/pBTdel-279-CD-TK on the 1st, 10th and 20th day after tumor implantation. In addition, the two kinds of plasmid (PEI/pBTdel-279-TK, PEI/pBTdel-279-CD-TK) treatment were also observed. Then 5-fluorocytosine (5-FC) & ganciclovir (GCV) were injected into the cavity of the peritoneum. The survival period and the survival prolong rate were calculated; (2) Human ovarian carcinoma SKOV3 cells were transfected by luciferase and the bioluminescent stability was evaluated in vitro. SKOV3-Luciferase stable cell line was implanted intraperitoneally to nude mice to establish the ovarian cancer animal model expressing luciferase. According to whether PEI/pBTdel-279-CD-TK therapy were given, the animals were divided into the treated model and the control model respectively. BLI monitoring of tumors growth and anticancer efficacy of suicide gene therapy in vivo were also applied timely.RESULTS: (1) The survival rate of tree different dose groups(5 μ g,30 μ g,50 μ g) were 25%,75% and 50% respectively. The average survival period of the treated group which pBTdel-279-CD-TK was injected on the 1st day after tumor
implantation was longer than other two groups (p<0.05). There were no significantly difference between the 10th day treated group and the 20th day treated group (P>0.05). The average survival period of the pBTdel-279-CD-TK group were prior to the pBTdel-279-TK group (P<0.05), and the survival prolong rate of the two groups were 146.44% and 73.41% respectively; (2) BLI showed that SKOV3-Luciferase cell line and its ovarian cancer intraperitoneal animal model expressing luciferase were established successfully. With the time passed, the luciferase signal gradually proliferated in the control model whereas it hardly could be detected in the treated model in vivo.CONCLUSION: The suicide gene therapy system using a human telomerase reverse transcnptase (hTERT) promoter by polyethylenimine mediated transfection had a significantly killing activity on human ovarian cancer in vivo; especially it had remarkably anti-tumor effects in the early period of the tumor development; Bioluminescent imaging could be used to monitor and evaluate ovarian cancer development and suicide gene therapy efficacy of PEI/hTERT-CD-TK more accurately and more sensitively, which indicated its potential to improve and refine traditional animal models.
第一部分 多聚乙烯基亚胺介导的卵巢特异性启动子调控下的自杀基因抑制卵巢癌生长的实验研究
目的:应用多聚乙烯基亚胺将卵巢特异性启动子OSP-1调控下的自杀基因HSV-tk导入人卵巢癌细胞,探讨其体外、体内的抗瘤效应和靶向性。
方法:(1)将pGL3-Luc质粒分别介导多聚乙烯基亚胺PEI、脂质体DOTAP和裸DNA转染人卵巢癌细胞SKOV3,检测荧光素酶表达RLU(relative luciferase unit);(2)利用PEI将卵巢特异性启动子调控下的真核表达质粒pOSP1-HSVtk
Recently with the rapid development of tumor gene therapy, the reseach on ovarian cancer has also become a hotspot. To improve the transfection efficiency and targeted delivery of gene vector have become two critical problems in the field of gene therapy. Many approaches have been developed to solve these problems. Polycationic polyethylenimine (PEI), as a new kind of nonviral gene vector, has provided an effective solution with its higher transfection efficiency and lower toxicity. The suicide gene therapy with a tumor specific promoter (hTERT) or an ovarian specific promoter (OSP1) is one of these targeted delivery strategies for ovarian cancer. In addition, establishing some relevant animal models is a necessary method in preclinical oncology research in order to observate tumor growth and effect before or after some treatment in the field of tumor gene therapy. However, the relevant animal models remain many question and challenge at present. Fortunately, bioluminescent imaging (BLI), as a new kind of animal model with its sensitive, rapid, less invasive and greater accurate, has provided a excellent technical solution. This subject aimed to investigate the transfection efficiency and targeted killing activity using specific suicide gene therapy system and polyethylenimine mediated transfection by establishing animal model of BLI for ovarian cancer. This subject was divided into two parts and the relevant abstract was as follows.
PART ⅠANTI-TUMOR STUDY OF SUICIDE GENE THERAPY WITH AN OVARIAN-SPECIFIC PROMOTER AND POLYETHYLENIMINE MEDIATED TRANSFECTION FOR OVARIAN CANCEROBJECTIVE: To investigate the anti-tumor and targeted delivery effects of HSV-tk suicide gene therapy adopting an ovarian-specific promoter (OSPl) for human ovarian carcinoma by polyethylenimine-mediated transfection in vitro and in vivo.METHODS: (1) Human ovarian carcinoma SKOV3 cells were transfected by pGL3-Luc plasmid mediated with polyethylenimine (PEI), DOTAP liposome and naked DNA respectively. The transfection efficiency was measured by RLU (relative luciferase unit); (2) The pOSPl-HSVtk plasmid containing an ovarian-specific promoter was transfected into the human ovarian carcinoma cell SKOV3 by PEI, human lung carcinoma cell NCI-H460 and human heptocellular carcinoma cell HepG2 and SMMC7721. The cytotoxicities resulted from GCV were evaluated using the MTT assay. The concentrations of GCV in SKOV3 cells were detected by high performance liquid chromatography (HPLC). The apoptosis of SKOV3 cells were observed by flow cytometry (FCM) and TdT-mediated dUTP nick end labeling (TUNEL) technique; (3) SKOV3 ovarian cancer xenograft model was established in BALB/C nude mice. The tumors were transfected with pOSPl-HSVtk using PEI as the transfection agent. The expressed TK activities were estimated by monitoring the GCV concentration change using a HPLC assay; (4) The weight of the nude mice, the tumor volumes and tumor weights were all recorded to calculate the tumor inhibition rates by weight and by volume. Histopathological analysis and TUNEL method were also performed; (5) SKOV3 cells were implanted intraperitoneally to BALB/C nude mice, then the survival period and survival prolong rate of BALB/C nude mice were evaluated the efficacy of PEI mediated pOSP1-HSVtk gene delivery followed by GCV treatment in vivo.RESULTS: (1)The polyethylenimine group had the highest luciferase activity
(187.35 ± 6.48) compared with other two groups (45.74±5.98, 0.03 ± 0.00) with significant statistical difference(P<0.01); (2) GCV was shown to be toxicity only in SKOV3 cells, not in NCI-H460 cells, SMMC7721and HepG2 cells. The concentrations of GCV in SKOV3 cells detected by HPLC gradually decreased with increase of time. FCM showed the apoptotic rate of cells significantly increased in SKOV3 exposed to GCV 24h, 48h and 72h (8.42 ±0.76%, 18.50±1.78%, 34.80+ 3.46%). The substantial cell apoptosis in SKOV3 was confirmed by TUNEL; (3) Compared with the control group in vivo, GCV concentration in tumor tissues transfected pOSP1-HSVtk were shown to be significantly lower by HPLC (0.05>P>0.01); (4) The tumor volume and the tumor weight in the treated group were also significantly decreased (P<0.01). The tumor volume inhibition rate and the tumor weight inhibition rate were estimated to be 63.66% and 58.98% respectively. Histological examination revealed heavy haemorrhage and necrosis in the tumor tissues, and TUNEL confirmed substantial cell apoptosis in the treated group; (5) Compared with these control groups, the survival period of the treated group was remarkably longer in the ovarian cancer survival model (P<0.01). The survival prolong rate of treated group was 63.80%.CONCLUSION: The suicide gene therapy system using an ovarian-specific promoter by polyethylenimine mediated transfection had a tissue-specific killing activity for human ovarian cancer in vitro and in vivo, which indicated its potential to improve the efficiency and the targeting in the field of gene therapy.
PART ⅡANTI-TUMOR EFFICACY STUDY OF DOUBLE SUICIDE GENETHERAPY WITH A hTERT PROMOTER FOR OVARIANCANCER BY POLYETHYLENIMINE MEDIATEDTRANSFECTIONOBJECTIVE: To evaluate the killing activity of double suicide gene therapy adopting a human telomerase reverse transcriptase (hTERT) promoter and polyethylenimine mediated transfection for human ovarian carcinoma in vivo by establishing traditional SKOV3 intraperitoneal implantation animal model as well as SKOV3-Luciferase intraperitoneal animal mode of bioluminescent imaging (BLI) respectively.METHODS: (1) SKOV3 cells were implanted intraperitoneally to BALB/C nude mice and the ovarian cancer survival model was established. Diverse dose (5 μ g, 30 μ g, 50 μ g) of pBTdel-279-CD-TK were given by intraperitoneal injection using PEI as the transfection agent. Diverse treatments were started by intraperitoneal injection PEI/pBTdel-279-CD-TK on the 1st, 10th and 20th day after tumor implantation. In addition, the two kinds of plasmid (PEI/pBTdel-279-TK, PEI/pBTdel-279-CD-TK) treatment were also observed. Then 5-fluorocytosine (5-FC) & ganciclovir (GCV) were injected into the cavity of the peritoneum. The survival period and the survival prolong rate were calculated; (2) Human ovarian carcinoma SKOV3 cells were transfected by luciferase and the bioluminescent stability was evaluated in vitro. SKOV3-Luciferase stable cell line was implanted intraperitoneally to nude mice to establish the ovarian cancer animal model expressing luciferase. According to whether PEI/pBTdel-279-CD-TK therapy were given, the animals were divided into the treated model and the control model respectively. BLI monitoring of tumors growth and anticancer efficacy of suicide gene therapy in vivo were also applied timely.RESULTS: (1) The survival rate of tree different dose groups(5 μ g,30 μ g,50 μ g) were 25%,75% and 50% respectively. The average survival period of the treated group which pBTdel-279-CD-TK was injected on the 1st day after tumor
implantation was longer than other two groups (p<0.05). There were no significantly difference between the 10th day treated group and the 20th day treated group (P>0.05). The average survival period of the pBTdel-279-CD-TK group were prior to the pBTdel-279-TK group (P<0.05), and the survival prolong rate of the two groups were 146.44% and 73.41% respectively; (2) BLI showed that SKOV3-Luciferase cell line and its ovarian cancer intraperitoneal animal model expressing luciferase were established successfully. With the time passed, the luciferase signal gradually proliferated in the control model whereas it hardly could be detected in the treated model in vivo.CONCLUSION: The suicide gene therapy system using a human telomerase reverse transcnptase (hTERT) promoter by polyethylenimine mediated transfection had a significantly killing activity on human ovarian cancer in vivo; especially it had remarkably anti-tumor effects in the early period of the tumor development; Bioluminescent imaging could be used to monitor and evaluate ovarian cancer development and suicide gene therapy efficacy of PEI/hTERT-CD-TK more accurately and more sensitively, which indicated its potential to improve and refine traditional animal models.
引文
1. Anderson WF. Human gene therapy. Science, 1992, 256: 808-813.
2. Cambron A. The Human Genome Project and the right to intellectual property, Law Hum Genome Rev, 2000, 7-12(13): 79-102.
3.Jain KK.Textbook of Gene Therapy(中文版).世界图书出版公司,2000,1:29-50.
4. Rochlitz CF. Gene therapy of cancer. Swiss Med Wkly, 2001, 131 (1-2): 4-9.
5. Fox JL. Gene therapy safety issues come to fore. Nat Biotech, 1999, 17(2): 1153-1157.
6. Li S, Ma Z. Nonviral gene therapy. Curr Gene Ther. 2001, 1(2): 201-226.
7. Mastrobattista E, Kapel RH, Eggenhuisen MH, et al. Lipid-coated polyplexes for targeted gene delivery to ovarian carcinoma cells. Cancer Gene Ther, 2001, 8(6): 405-413.
8. Schatzlein AG. Non-viral vectors in cancer gene therapy: principles and progress. Anticancer Drugs, 2001, 12(4): 275-304.
9. Brown MD, Schatzlein AG, Uchegbu IF. Gene delivery with synthetic (non viral) carders. Int J Phann, 2001, 229(1-2): 1-21.
10. Verma IM, Somia N. Gene therapy—promises, problems and prospects. Nature, 1997, 389(6648): 239-242.
11. Hill IR, Garnett MC, Bignotti F, et al. Determination of protection from serum nuclease activity by DNA-polyelectrolyte complexes using an electrophoretic method. Anal Bioehem, 2001, 291 (1): 62-68.
12. Gebhart CL, Kabanov AV. Evaluation of polyplexes as gene transfer agents. J Control Release, 2001, 73(2-3): 401-416.
13. Roy K, Mao HQ, Huang SK, et al. Oral gene delivery with ehitosan—DNA nanopartieles generates immunologic protection in a murine model of peanut allergy. Nat Med, 1999, 5(4): 387-391.
14. Legendre JY, Trzeciak A, Bur D, et al. N-acyl-(alpha, gamma diaminobutyric acid)n hydrazide as an efficient gene transfer vector in mammalian cells in culture. Pharm Res, 1997,14(5): 619-624.
15. Chemin I, Moradpour D, Wieland S, et al. Liver-directed gene transfer: a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo. J Viral Hepat, 1998, 5(6): 369-375.
16. Wu GY, Wu CH.Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem, 1987,262(10): 4429-4432.
17. Brown MD, Schatzlein A, Brownlie A, et al. Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents. Bioconjug Chem, 2000,11(6): 880-891.
18. Chen QR, Zhang L, Stass SA, et al. Branched co-polymers of histidine and lysine are efficient carriers of plasmids. Nucleic Acids Res, 2001,29(6): 1334-1340.
19. Hejazi R, Amiji M. Chitosan-based gastrointestinal delivery systems. J Control Release, 2003, 89(2): 151-165.
20. Kircheis R, Kichler A, Wallner G, et al. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther, 1997,4(5): 409-418.
21. Goula D, Remy JS, Erbacher P, et al. Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther, 1998,5(5): 712-717.
22. Poulain L, Ziller C, Muller CD,et al. Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives. Cancer Gene Ther, 2000,7(4):644-652.
23. Iwai M, Harada Y, Tanaka S, et al. Polyethylenimine-mediated suicide gene transfer induces a therapeutic effect for hepatocellular carcinoma in vivo by using an Epstein-Barr virus-based plasmid vector. Biochem Biophys Res Commun, 2002,291(1): 48-54.
24. Jeong JH, Kim SW, Park TG. A new antisense oligonucleotide delivery system based on self-assembled ODN-PEG hybrid conjugate micelles. J Control Release,
2003, 93(2): 183-191.
25. Kircheis R, Wightman L, Wagner E. Design and gene delivery activity of modified polyethylenimines. Adv Drug Deliv Rev, 2001, 53(3): 341-358.
26. Mastrobattista E, Crommelin D J, Wilschut J, et al. Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells. J Liposome Res, 2002, 12(1-2): 57-65.
27. Griffith GL, Edwards RT, Gray J. Cancer genetics services: a systematic review of the economic evidence and issues. Br J Cancer, 2004, 90(9): 1697-703.
28.贾林涛,王成济,杨安钢.肿瘤基因治疗的靶向性策略.中国癌症杂志,2003,13(2):174-177.
29. Spear MA. Gene therapy of gliomas: receptor and transcriptional targeting. Anticancer Res. 1998, 18(5A): 3223-3231.
30. Krasnykh VN, Douglas JT, van Beusechem VW. Genetic targeting of adenoviral vectors. Mol Ther, 2000, 1(5 Pt 1): 391-405.
31. Shimura H, Suzuki H, Miyazaki A, et al. Transcriptional activation of the thyroglobulin promoter directing suicide gene expression by thyroid transcription factor-1 in thyroid cancer cells. Cancer Res. 2001, 61(9): 3640-3646.
32. Tanyi JL, Lapushin R, Eder A, et al. Identification of tissue- and cancer-selective promoters for the introduction of genes into human ovarian cancer cells. Gynecol Oncol, 2002, 85(3): 451-458.
33. Poole JC, Andrews LG, Tollefsbol TO. Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT). Gene, 2001, 269(1-2): 1-12.
34. Hahn WC, Meyerson M. Telomerase activation, cellular immortalization and cancer. Ann Med, 2001, 33(2): 123-129.
35. Majumdar AS, Hughes DE, Lichtsteiner SP, et al. The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters. Gene Ther. 2001, 8(7): 568-578.
36. Kyo S, Inoue M. Complex regulatory mechanisms of telomerase activity in normal and cancer cells: how can we apply them for cancer therapy? Oncogene. 2002, 21 (4): 688-697.
37. Horikawa I, Cable PL, Mazur SJ, et al. Downstream E-box-mediated regulation of the human telomerase revers transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression. Mol Biol Cell, 2002, 13(8): 2585-2597.
38. Braunstein I, Cohen-Barak O, Shachaf C, et al. Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells. Cancer Res, 2001, 61(14): 5529-5536.
39. Berry NB, Cho YM, Harrington MA, et al. Transcriptional targeting in ovarian cancer cells using the human epididymis protein 4 promoter. Gynecol Oncol, 2004, 92(3): 896-904.
40. Anderson LM, Swaminathan S, Zackon I, et al. Adenovirus-mediated tissue-targeted expression of the HSVtk gene for the treatment of breast cancer. Gene Ther. 1999, 6(5): 854-864.
41. Lee S J, Kim HS, Yu R, et al. Novel prostate-specific promoter derived from PSA and PSMA enhancers, Mol Ther, 2002, 6(3): 415-421.
42. Hou Z, Nguyen Q, Frenkel B, et al. Osteoblast-specific gene expression after transplantation of marrow cells: implications for skeletal gene therapy. Proc Natl Acad Sci U S A, 1999, 96(13): 7294-7299.
43. Chen J, Bezdek T, Chang J, et al. A glial-specific, repressible, adenovirus vector for brain tumor gene therapy. Cancer Res, 1998, 58(16): 3504-3507.
44. Li S, MaeLaughlin FC, Fewell JG, et al. Increased level and duration of expression in muscle by co-expression of a transactivator using plasmid systems. Gene Ther, 1999, 6(12): 2005-2011.
45. Selvakumaran M, Bao R, Crijns AP, et al. Ovarian epithelial cell lineage-specific gene expression using the promoter of a retrovirus-like element. Cancer Res, 2001, 61(4): 1291-1295.
46.吴细丕,钱林法主编.实验动物与肿瘤研究.北京:中国医药科技出版 社.2000,18-19.
47. Jenkins DE, Oei Y, Hornig YS, et al. Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis. Clinical & Experimental Metastasis, 2003, 20: 733-744.
48.张龙江,宋光义.分子成像的研究进展.国外医学临床放射学分册,2002,25(5):266—268.
49.孙敬方主编.动物实验方法学.北京:人民卫生出版社.2001,513—527.
50. Weissleder R. Scaling down imaging: Molecular mapping of cancer in mice. Nat Rev Cancer, 2002, 2(1): 11-18.
51. Gambhir SS. Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer, 2002, 2(9): 683-693.
52. Contag PR. Whole-animal cellular and molecular imaging to accelerate drug development. Drug Discov Today, 2002, 7(10): 555-562.
53. Edinger M, Hoffmann P, Contag CH, et al. Evaluation of effector cell fate and function by in vivo bioluminescence imaging. Methods, 2003, 31 (2): 172-179.
54. Zhang G J, Safran M, Wei W, et al. Bioluminescent imaging of Cdk2 inhibition in vivo. Nat Med, 2004, 10 (6): 643-648.
55. Sato A, Klaunberg B, Tolwani R. In vivo bioluminescence imaging. Comp Med, 2004, 54 (6): 631-634.
56. Hoffman R. Green fluorescent protein imaging of tumor growth, metastasis, and angiogenesis in mouse models. Lancet Oncol, 2002, 3(9): 546-556.
57. Weissleder R, Ntziachristos V. Shedding light onto live molecular target. Nat Med, 2003, 9(1): 123-128.
58. Hollingshead MG, Bonomi CA, Borgel SD, et al. A potential role for imaging technology in anticancer efficacy evaluations. Eur J Cancer, 2004, 40(6): 890-898.
59. Luker GD, Leib DA. Luciferase real-time bioluminescence imaging for the study of viral pathogenesis. Methods Mol Biol, 2005; 292: 285-296.
60. Jawhara S, Mordon S. In vivo imaging of bioluminescent Escherichia coli in a cutaneous wound infection model for evaluation of an antibiotic therapy. Antimicrob Agents Chemother, 2004, 48(9): 3436-3441.
61. Rocchetta HL, Boylan C J, Foley JW, et al. Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection. Antimicrob Agents Chemother, 2001, 45(1): 129-137.
62. Kadurugamuwa JL, Sin LV, Yu J, et al. Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo. Antimicrob Agents Chemother, 2004, 48(6): 2283-2287.
63. Kuklin NA, Pancari GD, Tobery TW, et al. Real-time monitoring of bacterial infection in vivo: development of bioluminescent staphylococcal foreign-body and deep-thigh-wound mouse infection models. Antimicrob Agents Chemother, 2003, 47(9): 2740-2748.
64. Carlsen H, Alexander G, Austenaa LM, et al. Molecular imaging of the transcription factor NF-kappaB, a primary regulator of stress response. Murat Res, 2004, 551(1-2): 199-211.
65. Iris B, Zilberman Y, Zeira E, et al. Molecular imaging of the skeleton: quantitative real-time bioluminescence monitoring gene expression in bone repair and development. J Bone Miner Res, 2003, 18(3): 570-578.
66. Honigman A, Zeira E, Ohana P, et al. Imaging transgene expression in live animals. Mol Ther, 2001, 4(3): 239-249.
67. Qian C, Idoate M, Bilbao R, et al. Gene transfer and therapy with adenoviral vector in rats with diethylnitrosamine-induced hepatocellular carcinoma. Hum Gene Ther, 1997, 8(3): 349-358.
68.司徒镇强,吴军正主编.细胞培养.北京:世界图书出版社.1996,73-83.
69.Sambmok J,Fritsh EF,Kingston T,等著.分子克隆实验指南.第二版.金冬雁等译.北京:科学出版社.1992,956.
70.张均田主编.现代药理实验方法.北京:北京医科大学中国协和医科大学联合出版社.1607-1611.
71. Haberkom U, Khazaie K, Morr I, et al. Ganciclovir uptake in human mammary carcinoma cells expressing herpes simplex virus thymidine kinase. Nucl Med Biol, 1998, 25(4): 367-373.
72. Xu K, Lanuti M, Lambright ES, et al. A rapid and sensitive method for the quantification of ganciclovir in plasma using liquid chromatography/selected reaction monitoring/mass spectrometry. Biomed Chromatogr, 2000, 14: 93-98.
73.黄洪章,王安训,李苏,等.HSV-TK/GCV系统治疗口腔鳞癌移植瘤的研究.中山医科大学学报,2002,23(5):372-374.
74. Bao R, Selvakumaran M, Hamilton TC. Targeted gene therapy of ovarian cancer using an ovarian-specific promoter. Gynecol Oncol, 2002, 84(2): 228-234.
75. Ozols RF. Future directions in the treatment of ovarian cancer. Semin Oncol, 2002, 29(1 Suppl 1): 32-42.
76. Peethambaram PP, Long HJ. Second-line and subsequent therapy for ovarian carcinoma. Curr Oncol Rep, 2002, 4(2): 159-164.
77. DiSaia P J, Bloss JD. Treatment of ovarian cancer: new strategies. Gynecol Oncol. 2003, 90(2 Pt 2): S24-32.
78. Casado E, Nettelbeck DM, Gomez-Navarro J, et al. Transcriptional targeting for ovarian cancer gene therapy, Gynecol Oncol, 2001, 82(2): 229-237.
79. Barnes MN, Coolidge C J, Hemminki A, et al. Conditionally replicative adenoviruses for ovarian cancer therapy. Mol Cancer Ther, 2002, 1 (6): 435-439.
80. Wolf JK, Jenkins AD. Gene therapy for ovarian cancer (review). Int J Oncol, 2002, 21(3): 461-468.
81. Hermiston T. Gene delivery from replication-selective viruses: arming guided missiles in the war against cancer. J Clin Invest, 2000, 105(9): 1169-1172.
82. Link CJ Jr, Moorman D, Seregina T, et al. A phase I trial of in vivo gene therapy with the herpes simplex thymidine kinase/ganciclovir system for the treatment of refractory or recurrent ovarian cancer. Hum Gene Ther. 1996, 7(9): 1161-1179.
83. Alvarez RD, Gomez-Navarro J, Wang M, et al. Adenoviral-mediated suicide gene therapy for ovarian cancer. Mol Ther, 2000, 2(5): 524-530.
84.童荣生.非病毒基因治疗载体的研究进展.中国药学杂志,2004,39(1):1-4.
85. Boussif O, Lezoualc'h F, Zanta MA, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A, 1995, 92(16): 7297-7301.
86. Kichler A, Leborgne C, Coeytaux E, et al. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med, 2001, 3(2): 135-144.
87. Boussif O, Zanta MA, Behr JP. Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther, 1996, 3 (12): 1074-1080.
88. Oh YK, Suh D, Kim JM, et al. Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Ther, 2002, 9(23): 1627-1632.
89. Dunlap DD, Maggi A, Soria MR, et al. Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acids Res, 1997, 25 (15): 3095-3101.
90. Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability. Biomaterials, 2001, 22(5): 471-480.
91. Poulain L, Ziller C, Muller CD, et al. Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives. Cancer Gene Ther, 2000, 7(4): 644-652.
92. Forrest ML, Koerber JT, Pack DW. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug Chem, 2003, 14(5): 934-940.
93. Choosakoonkriang S, Lobo BA, Koe GS, et al. Biophysical characterization of PEI/DNA complexes, J Pharm Sci, 2003, 92(8): 1710-1722.
94. Pouton CW, Seymour LW. Key issues in non-viral gene delivery. Adv Drug Deliv Rev, 2001, 46(1-3): 187-203.
95. Ross PC, Hui SW. Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Ther, 1999, 6(4): 651-659.
96. Guo W, Lee RJ. Efficient gene delivery via non-covalent complexes of folio acid and polyethylenimine. J Control Release, 2001, 77(1-2): 131-138.
97.李经忠,王青青,曹雪涛.新型非病毒载体聚乙烯亚胺体内应用的研究进展.国外医学药学分册,2004,31(1):34-37.
98. Goula D, Benoist C, Mantero S, et al. Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther, 1998, 5(9): 1291-1295.
99. Goula D, Becker N, Lemkine GF, et al. Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes. Gene Ther, 2000, 7(6): 499-504.
100. Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Ther, 2000, 7(1): 31-34.
101. Kircheis R, Schuller S, Brunner S, et al. Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. J Gene Med, 1999, 1(2): 111-120.
102. Dolivet G, Merlin JL, Barberi-Heyob M, et al. In vivo growth inhibitory effect of iterative wild-type p53 gene transfer in human head and neck carcinoma xenografts using glucosylated polyethylenimine nonviral vector. Cancer Gene Ther, 2002, 9(8): 708-714.
103. Zou SM, Erbacher P, Remy JS, et al. Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse. J Gene Med, 2000, 2(2): 128-134.
104. Boletta A, Benigni A, Lutz J, et al. Nonviral gene delivery to the rat kidney with polyethylenimine. Hum Gene Ther, 1997, 8(10): 1243-1251.
105. Coll JL, Chollet P, Brambilla E,. et al. In vivo delivery to tumors of DNA complexed with linear polyethylenimine. Hum Gene Ther, 1999, 10(10): 1659-1666.
106.陈建海.阳离子聚合物在基因传递系统中的应用.药学学报,2003,38(4):316-320.
107. Lee CH, Ni YH, Chen CC, et al. Synergistic effect of polyethylenimine and cationic liposomes in nucleic acid delivery to human cancer cells. Biochim Biophys Acta, 2003, 1611 (1-2): 55-62.
108. Jeong JH, Kim SW, Park TG. A new antisense oligonucleotide delivery system based on self-assembled ODN-PEG hybrid conjugate micelles. J Control Release, 2003, 93(2): 183-191.
109. Nishihara E, Nagayama Y, Narimatsu M, et al. Treatment of thyroid carcinoma cells with four different suicide gene/prodrug combinations in vitro. Anticancer Res, 1998, 18(3A): 1521-1525.
110. Uckert W, Kammertons T, Haack K, et al. Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo. Hum Gene Ther, 1998, 9(6): 855-865.
111.陈道桢,张丽珊.自杀基因治疗肿瘤研究进展.国外医学分子生物学分册,2002,24(1):23-26.
112. Moolten FL, Wells JM, Heyman RA, et al. Lymphoma regression induced by ganciclovir in mice beating a herpes thymidine kinase transgene. Hum Gene Ther, 1990, 1(2): 125-134.
113. Desaknai S, Lumniczky K, Esik O, et al. Local tumour irradiation enhances the anti-tumour effect of a double-suicide gene therapy system in a murine glioma model. J Gene Med, 2003, 5(5): 377-385.
114. Shand N, Weber F, Mariani L, et al. A phase 1-2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir. GLI328 European-Canadian Study Group.Hum Gene Ther, 1999, 10(14): 2325-2335.
115.孔北华,宋悦,马道新,等.人端粒酶逆转录酶启动子调控下胞嘧啶脱氨酶-胸苷激酶融合基因治疗卵巢癌的体外研究.中华妇产科杂志,2004,6(39):390-395.
116.路春华,盛修贵.卵巢上皮癌自杀基因疗法研究进展.肿瘤学杂志,2004,10(2):107-109.
117. Kim JH, Kolozsvary A, Rogulski K, et al. Selective radiosensitization of 9L glioma in the brain transduced with double suicide fusion gene. Cancer J Sci Am, 1998, 4(6): 364-369.
118. Rogulski KR, Zhang K, Kolozsvary A, et al. Pronounced antitumor effects and tumor radiosensitization of double suicide gene therapy. Clin Cancer Res, 1997, 3(11): 2081-2088.
119. Consalvo M, Mullen CA, Modesti A, et al. 5-Fluorocytosine-induced eradication of murine adenocarcinomas engineered to express the cytosine deaminase suicide gene requires host immune competence and leaves an efficient memory. J Immunol, 1995, 154(10): 5302-5312.
120. Szala S, Missol E, Sochanik A, et al. The use of cationic liposomes DC-CHOL/DOPE and DDAB/DOPE for direct transfer of Escherichia coli cytosine deaminase gene into growing melanoma tumors. Gene Ther, 1996, 3(11): 1026-1031.
121. Bruce JH, Harvey LN. Translating molecular imaging research into radiologic practice. Radiology, 2002, 222: 19-24.
122. O' Connell-Rodwell CE, Bums SM, Bachmann MH, et al. Bioluminescent indicators for in vivo measurements of gene expression. Trends in Biotechnology, 2002, 20(8Suppl.): S19-S23.
123. Freeman SM, Ramesh R, Marrogi AJ. Immune system in suicide-gene therapy. Lancet, 1997, 349(9044): 2-3.
124. Yamamoto S, Suzuki S, Hoshino A, et al. Herpes simplex virus thymidine kinase/ganciclovir-mediated killing of tumor cell induces tumor-specific cytotoxic T cells in mice. Cancer Gene Ther, 1997, 4(2): 91-96.
125. Haack K, Linnebacher M, Eisold S, et al. Induction of protective immunity against syngeneic rat cancer cells by expression of the cytosine deaminase suicide gene. Cancer Gene Ther, 2000, 7(10): 1357-1364.
126. Gavelli A, Baque P, Mala M, et al. Vaccination by suicide gene therapy against a model of hepatic metastasis from colon cancer in the rat. Ann Chir, 2000, 125(6): 552-559.
127. Nagy H J, Panis Y, Fabre M, et al. Efficient suicide gene therapy of transduced and distant untransduced ovary, tumors is correlated with significant increase of intratumoral T and NK cells. Biomed Pharmacother, 2000, 54(10): 479-486.
128.罗满林,顾为望主编.实验动物学.北京:中国农业出版社.2002,113-114.
129.Roda A, Pasini P, Mirasoli M, et al. Biotechnological applications of bioluminescence and chemiluminescence. Trends Biotechnol, 2004,22(6): 295-303.
130.Paroo Z, Bollinger RA, Braasch DA, et al. Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden. Mol Imaging, 2004,3(2): 117-124.
131.Shah K, Jacobs A, Breakefield XO, et al. Molecular imaging of gene therapy for cancer. Gene Ther. 2004, 11(15): 1175-1187.
132.Iyer M, Salazar FB, Lewis X, et al. Noninvasive imaging of enhanced prostate-specific gene expression using a two-step transcriptional amplification-based lentivirus vector. Mol Ther, 2004,10(3): 545-552.
133.Uhrbom L, Nerio E, Holland EC. Dissecting tumor maintenance requirements using bioluminescence imaging of cell proliferation in a mouse glioma model. Nat Med, 2004,10(11): 1257-1260.
134.Lyons SK. Advances in imaging mouse tumour models in vivo. J Pathol, 2005, 205(2): 194-205.
2. Cambron A. The Human Genome Project and the right to intellectual property, Law Hum Genome Rev, 2000, 7-12(13): 79-102.
3.Jain KK.Textbook of Gene Therapy(中文版).世界图书出版公司,2000,1:29-50.
4. Rochlitz CF. Gene therapy of cancer. Swiss Med Wkly, 2001, 131 (1-2): 4-9.
5. Fox JL. Gene therapy safety issues come to fore. Nat Biotech, 1999, 17(2): 1153-1157.
6. Li S, Ma Z. Nonviral gene therapy. Curr Gene Ther. 2001, 1(2): 201-226.
7. Mastrobattista E, Kapel RH, Eggenhuisen MH, et al. Lipid-coated polyplexes for targeted gene delivery to ovarian carcinoma cells. Cancer Gene Ther, 2001, 8(6): 405-413.
8. Schatzlein AG. Non-viral vectors in cancer gene therapy: principles and progress. Anticancer Drugs, 2001, 12(4): 275-304.
9. Brown MD, Schatzlein AG, Uchegbu IF. Gene delivery with synthetic (non viral) carders. Int J Phann, 2001, 229(1-2): 1-21.
10. Verma IM, Somia N. Gene therapy—promises, problems and prospects. Nature, 1997, 389(6648): 239-242.
11. Hill IR, Garnett MC, Bignotti F, et al. Determination of protection from serum nuclease activity by DNA-polyelectrolyte complexes using an electrophoretic method. Anal Bioehem, 2001, 291 (1): 62-68.
12. Gebhart CL, Kabanov AV. Evaluation of polyplexes as gene transfer agents. J Control Release, 2001, 73(2-3): 401-416.
13. Roy K, Mao HQ, Huang SK, et al. Oral gene delivery with ehitosan—DNA nanopartieles generates immunologic protection in a murine model of peanut allergy. Nat Med, 1999, 5(4): 387-391.
14. Legendre JY, Trzeciak A, Bur D, et al. N-acyl-(alpha, gamma diaminobutyric acid)n hydrazide as an efficient gene transfer vector in mammalian cells in culture. Pharm Res, 1997,14(5): 619-624.
15. Chemin I, Moradpour D, Wieland S, et al. Liver-directed gene transfer: a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo. J Viral Hepat, 1998, 5(6): 369-375.
16. Wu GY, Wu CH.Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem, 1987,262(10): 4429-4432.
17. Brown MD, Schatzlein A, Brownlie A, et al. Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents. Bioconjug Chem, 2000,11(6): 880-891.
18. Chen QR, Zhang L, Stass SA, et al. Branched co-polymers of histidine and lysine are efficient carriers of plasmids. Nucleic Acids Res, 2001,29(6): 1334-1340.
19. Hejazi R, Amiji M. Chitosan-based gastrointestinal delivery systems. J Control Release, 2003, 89(2): 151-165.
20. Kircheis R, Kichler A, Wallner G, et al. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther, 1997,4(5): 409-418.
21. Goula D, Remy JS, Erbacher P, et al. Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther, 1998,5(5): 712-717.
22. Poulain L, Ziller C, Muller CD,et al. Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives. Cancer Gene Ther, 2000,7(4):644-652.
23. Iwai M, Harada Y, Tanaka S, et al. Polyethylenimine-mediated suicide gene transfer induces a therapeutic effect for hepatocellular carcinoma in vivo by using an Epstein-Barr virus-based plasmid vector. Biochem Biophys Res Commun, 2002,291(1): 48-54.
24. Jeong JH, Kim SW, Park TG. A new antisense oligonucleotide delivery system based on self-assembled ODN-PEG hybrid conjugate micelles. J Control Release,
2003, 93(2): 183-191.
25. Kircheis R, Wightman L, Wagner E. Design and gene delivery activity of modified polyethylenimines. Adv Drug Deliv Rev, 2001, 53(3): 341-358.
26. Mastrobattista E, Crommelin D J, Wilschut J, et al. Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells. J Liposome Res, 2002, 12(1-2): 57-65.
27. Griffith GL, Edwards RT, Gray J. Cancer genetics services: a systematic review of the economic evidence and issues. Br J Cancer, 2004, 90(9): 1697-703.
28.贾林涛,王成济,杨安钢.肿瘤基因治疗的靶向性策略.中国癌症杂志,2003,13(2):174-177.
29. Spear MA. Gene therapy of gliomas: receptor and transcriptional targeting. Anticancer Res. 1998, 18(5A): 3223-3231.
30. Krasnykh VN, Douglas JT, van Beusechem VW. Genetic targeting of adenoviral vectors. Mol Ther, 2000, 1(5 Pt 1): 391-405.
31. Shimura H, Suzuki H, Miyazaki A, et al. Transcriptional activation of the thyroglobulin promoter directing suicide gene expression by thyroid transcription factor-1 in thyroid cancer cells. Cancer Res. 2001, 61(9): 3640-3646.
32. Tanyi JL, Lapushin R, Eder A, et al. Identification of tissue- and cancer-selective promoters for the introduction of genes into human ovarian cancer cells. Gynecol Oncol, 2002, 85(3): 451-458.
33. Poole JC, Andrews LG, Tollefsbol TO. Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT). Gene, 2001, 269(1-2): 1-12.
34. Hahn WC, Meyerson M. Telomerase activation, cellular immortalization and cancer. Ann Med, 2001, 33(2): 123-129.
35. Majumdar AS, Hughes DE, Lichtsteiner SP, et al. The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters. Gene Ther. 2001, 8(7): 568-578.
36. Kyo S, Inoue M. Complex regulatory mechanisms of telomerase activity in normal and cancer cells: how can we apply them for cancer therapy? Oncogene. 2002, 21 (4): 688-697.
37. Horikawa I, Cable PL, Mazur SJ, et al. Downstream E-box-mediated regulation of the human telomerase revers transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression. Mol Biol Cell, 2002, 13(8): 2585-2597.
38. Braunstein I, Cohen-Barak O, Shachaf C, et al. Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells. Cancer Res, 2001, 61(14): 5529-5536.
39. Berry NB, Cho YM, Harrington MA, et al. Transcriptional targeting in ovarian cancer cells using the human epididymis protein 4 promoter. Gynecol Oncol, 2004, 92(3): 896-904.
40. Anderson LM, Swaminathan S, Zackon I, et al. Adenovirus-mediated tissue-targeted expression of the HSVtk gene for the treatment of breast cancer. Gene Ther. 1999, 6(5): 854-864.
41. Lee S J, Kim HS, Yu R, et al. Novel prostate-specific promoter derived from PSA and PSMA enhancers, Mol Ther, 2002, 6(3): 415-421.
42. Hou Z, Nguyen Q, Frenkel B, et al. Osteoblast-specific gene expression after transplantation of marrow cells: implications for skeletal gene therapy. Proc Natl Acad Sci U S A, 1999, 96(13): 7294-7299.
43. Chen J, Bezdek T, Chang J, et al. A glial-specific, repressible, adenovirus vector for brain tumor gene therapy. Cancer Res, 1998, 58(16): 3504-3507.
44. Li S, MaeLaughlin FC, Fewell JG, et al. Increased level and duration of expression in muscle by co-expression of a transactivator using plasmid systems. Gene Ther, 1999, 6(12): 2005-2011.
45. Selvakumaran M, Bao R, Crijns AP, et al. Ovarian epithelial cell lineage-specific gene expression using the promoter of a retrovirus-like element. Cancer Res, 2001, 61(4): 1291-1295.
46.吴细丕,钱林法主编.实验动物与肿瘤研究.北京:中国医药科技出版 社.2000,18-19.
47. Jenkins DE, Oei Y, Hornig YS, et al. Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis. Clinical & Experimental Metastasis, 2003, 20: 733-744.
48.张龙江,宋光义.分子成像的研究进展.国外医学临床放射学分册,2002,25(5):266—268.
49.孙敬方主编.动物实验方法学.北京:人民卫生出版社.2001,513—527.
50. Weissleder R. Scaling down imaging: Molecular mapping of cancer in mice. Nat Rev Cancer, 2002, 2(1): 11-18.
51. Gambhir SS. Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer, 2002, 2(9): 683-693.
52. Contag PR. Whole-animal cellular and molecular imaging to accelerate drug development. Drug Discov Today, 2002, 7(10): 555-562.
53. Edinger M, Hoffmann P, Contag CH, et al. Evaluation of effector cell fate and function by in vivo bioluminescence imaging. Methods, 2003, 31 (2): 172-179.
54. Zhang G J, Safran M, Wei W, et al. Bioluminescent imaging of Cdk2 inhibition in vivo. Nat Med, 2004, 10 (6): 643-648.
55. Sato A, Klaunberg B, Tolwani R. In vivo bioluminescence imaging. Comp Med, 2004, 54 (6): 631-634.
56. Hoffman R. Green fluorescent protein imaging of tumor growth, metastasis, and angiogenesis in mouse models. Lancet Oncol, 2002, 3(9): 546-556.
57. Weissleder R, Ntziachristos V. Shedding light onto live molecular target. Nat Med, 2003, 9(1): 123-128.
58. Hollingshead MG, Bonomi CA, Borgel SD, et al. A potential role for imaging technology in anticancer efficacy evaluations. Eur J Cancer, 2004, 40(6): 890-898.
59. Luker GD, Leib DA. Luciferase real-time bioluminescence imaging for the study of viral pathogenesis. Methods Mol Biol, 2005; 292: 285-296.
60. Jawhara S, Mordon S. In vivo imaging of bioluminescent Escherichia coli in a cutaneous wound infection model for evaluation of an antibiotic therapy. Antimicrob Agents Chemother, 2004, 48(9): 3436-3441.
61. Rocchetta HL, Boylan C J, Foley JW, et al. Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection. Antimicrob Agents Chemother, 2001, 45(1): 129-137.
62. Kadurugamuwa JL, Sin LV, Yu J, et al. Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo. Antimicrob Agents Chemother, 2004, 48(6): 2283-2287.
63. Kuklin NA, Pancari GD, Tobery TW, et al. Real-time monitoring of bacterial infection in vivo: development of bioluminescent staphylococcal foreign-body and deep-thigh-wound mouse infection models. Antimicrob Agents Chemother, 2003, 47(9): 2740-2748.
64. Carlsen H, Alexander G, Austenaa LM, et al. Molecular imaging of the transcription factor NF-kappaB, a primary regulator of stress response. Murat Res, 2004, 551(1-2): 199-211.
65. Iris B, Zilberman Y, Zeira E, et al. Molecular imaging of the skeleton: quantitative real-time bioluminescence monitoring gene expression in bone repair and development. J Bone Miner Res, 2003, 18(3): 570-578.
66. Honigman A, Zeira E, Ohana P, et al. Imaging transgene expression in live animals. Mol Ther, 2001, 4(3): 239-249.
67. Qian C, Idoate M, Bilbao R, et al. Gene transfer and therapy with adenoviral vector in rats with diethylnitrosamine-induced hepatocellular carcinoma. Hum Gene Ther, 1997, 8(3): 349-358.
68.司徒镇强,吴军正主编.细胞培养.北京:世界图书出版社.1996,73-83.
69.Sambmok J,Fritsh EF,Kingston T,等著.分子克隆实验指南.第二版.金冬雁等译.北京:科学出版社.1992,956.
70.张均田主编.现代药理实验方法.北京:北京医科大学中国协和医科大学联合出版社.1607-1611.
71. Haberkom U, Khazaie K, Morr I, et al. Ganciclovir uptake in human mammary carcinoma cells expressing herpes simplex virus thymidine kinase. Nucl Med Biol, 1998, 25(4): 367-373.
72. Xu K, Lanuti M, Lambright ES, et al. A rapid and sensitive method for the quantification of ganciclovir in plasma using liquid chromatography/selected reaction monitoring/mass spectrometry. Biomed Chromatogr, 2000, 14: 93-98.
73.黄洪章,王安训,李苏,等.HSV-TK/GCV系统治疗口腔鳞癌移植瘤的研究.中山医科大学学报,2002,23(5):372-374.
74. Bao R, Selvakumaran M, Hamilton TC. Targeted gene therapy of ovarian cancer using an ovarian-specific promoter. Gynecol Oncol, 2002, 84(2): 228-234.
75. Ozols RF. Future directions in the treatment of ovarian cancer. Semin Oncol, 2002, 29(1 Suppl 1): 32-42.
76. Peethambaram PP, Long HJ. Second-line and subsequent therapy for ovarian carcinoma. Curr Oncol Rep, 2002, 4(2): 159-164.
77. DiSaia P J, Bloss JD. Treatment of ovarian cancer: new strategies. Gynecol Oncol. 2003, 90(2 Pt 2): S24-32.
78. Casado E, Nettelbeck DM, Gomez-Navarro J, et al. Transcriptional targeting for ovarian cancer gene therapy, Gynecol Oncol, 2001, 82(2): 229-237.
79. Barnes MN, Coolidge C J, Hemminki A, et al. Conditionally replicative adenoviruses for ovarian cancer therapy. Mol Cancer Ther, 2002, 1 (6): 435-439.
80. Wolf JK, Jenkins AD. Gene therapy for ovarian cancer (review). Int J Oncol, 2002, 21(3): 461-468.
81. Hermiston T. Gene delivery from replication-selective viruses: arming guided missiles in the war against cancer. J Clin Invest, 2000, 105(9): 1169-1172.
82. Link CJ Jr, Moorman D, Seregina T, et al. A phase I trial of in vivo gene therapy with the herpes simplex thymidine kinase/ganciclovir system for the treatment of refractory or recurrent ovarian cancer. Hum Gene Ther. 1996, 7(9): 1161-1179.
83. Alvarez RD, Gomez-Navarro J, Wang M, et al. Adenoviral-mediated suicide gene therapy for ovarian cancer. Mol Ther, 2000, 2(5): 524-530.
84.童荣生.非病毒基因治疗载体的研究进展.中国药学杂志,2004,39(1):1-4.
85. Boussif O, Lezoualc'h F, Zanta MA, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A, 1995, 92(16): 7297-7301.
86. Kichler A, Leborgne C, Coeytaux E, et al. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med, 2001, 3(2): 135-144.
87. Boussif O, Zanta MA, Behr JP. Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther, 1996, 3 (12): 1074-1080.
88. Oh YK, Suh D, Kim JM, et al. Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Ther, 2002, 9(23): 1627-1632.
89. Dunlap DD, Maggi A, Soria MR, et al. Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acids Res, 1997, 25 (15): 3095-3101.
90. Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability. Biomaterials, 2001, 22(5): 471-480.
91. Poulain L, Ziller C, Muller CD, et al. Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives. Cancer Gene Ther, 2000, 7(4): 644-652.
92. Forrest ML, Koerber JT, Pack DW. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug Chem, 2003, 14(5): 934-940.
93. Choosakoonkriang S, Lobo BA, Koe GS, et al. Biophysical characterization of PEI/DNA complexes, J Pharm Sci, 2003, 92(8): 1710-1722.
94. Pouton CW, Seymour LW. Key issues in non-viral gene delivery. Adv Drug Deliv Rev, 2001, 46(1-3): 187-203.
95. Ross PC, Hui SW. Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Ther, 1999, 6(4): 651-659.
96. Guo W, Lee RJ. Efficient gene delivery via non-covalent complexes of folio acid and polyethylenimine. J Control Release, 2001, 77(1-2): 131-138.
97.李经忠,王青青,曹雪涛.新型非病毒载体聚乙烯亚胺体内应用的研究进展.国外医学药学分册,2004,31(1):34-37.
98. Goula D, Benoist C, Mantero S, et al. Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther, 1998, 5(9): 1291-1295.
99. Goula D, Becker N, Lemkine GF, et al. Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes. Gene Ther, 2000, 7(6): 499-504.
100. Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Ther, 2000, 7(1): 31-34.
101. Kircheis R, Schuller S, Brunner S, et al. Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. J Gene Med, 1999, 1(2): 111-120.
102. Dolivet G, Merlin JL, Barberi-Heyob M, et al. In vivo growth inhibitory effect of iterative wild-type p53 gene transfer in human head and neck carcinoma xenografts using glucosylated polyethylenimine nonviral vector. Cancer Gene Ther, 2002, 9(8): 708-714.
103. Zou SM, Erbacher P, Remy JS, et al. Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse. J Gene Med, 2000, 2(2): 128-134.
104. Boletta A, Benigni A, Lutz J, et al. Nonviral gene delivery to the rat kidney with polyethylenimine. Hum Gene Ther, 1997, 8(10): 1243-1251.
105. Coll JL, Chollet P, Brambilla E,. et al. In vivo delivery to tumors of DNA complexed with linear polyethylenimine. Hum Gene Ther, 1999, 10(10): 1659-1666.
106.陈建海.阳离子聚合物在基因传递系统中的应用.药学学报,2003,38(4):316-320.
107. Lee CH, Ni YH, Chen CC, et al. Synergistic effect of polyethylenimine and cationic liposomes in nucleic acid delivery to human cancer cells. Biochim Biophys Acta, 2003, 1611 (1-2): 55-62.
108. Jeong JH, Kim SW, Park TG. A new antisense oligonucleotide delivery system based on self-assembled ODN-PEG hybrid conjugate micelles. J Control Release, 2003, 93(2): 183-191.
109. Nishihara E, Nagayama Y, Narimatsu M, et al. Treatment of thyroid carcinoma cells with four different suicide gene/prodrug combinations in vitro. Anticancer Res, 1998, 18(3A): 1521-1525.
110. Uckert W, Kammertons T, Haack K, et al. Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo. Hum Gene Ther, 1998, 9(6): 855-865.
111.陈道桢,张丽珊.自杀基因治疗肿瘤研究进展.国外医学分子生物学分册,2002,24(1):23-26.
112. Moolten FL, Wells JM, Heyman RA, et al. Lymphoma regression induced by ganciclovir in mice beating a herpes thymidine kinase transgene. Hum Gene Ther, 1990, 1(2): 125-134.
113. Desaknai S, Lumniczky K, Esik O, et al. Local tumour irradiation enhances the anti-tumour effect of a double-suicide gene therapy system in a murine glioma model. J Gene Med, 2003, 5(5): 377-385.
114. Shand N, Weber F, Mariani L, et al. A phase 1-2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir. GLI328 European-Canadian Study Group.Hum Gene Ther, 1999, 10(14): 2325-2335.
115.孔北华,宋悦,马道新,等.人端粒酶逆转录酶启动子调控下胞嘧啶脱氨酶-胸苷激酶融合基因治疗卵巢癌的体外研究.中华妇产科杂志,2004,6(39):390-395.
116.路春华,盛修贵.卵巢上皮癌自杀基因疗法研究进展.肿瘤学杂志,2004,10(2):107-109.
117. Kim JH, Kolozsvary A, Rogulski K, et al. Selective radiosensitization of 9L glioma in the brain transduced with double suicide fusion gene. Cancer J Sci Am, 1998, 4(6): 364-369.
118. Rogulski KR, Zhang K, Kolozsvary A, et al. Pronounced antitumor effects and tumor radiosensitization of double suicide gene therapy. Clin Cancer Res, 1997, 3(11): 2081-2088.
119. Consalvo M, Mullen CA, Modesti A, et al. 5-Fluorocytosine-induced eradication of murine adenocarcinomas engineered to express the cytosine deaminase suicide gene requires host immune competence and leaves an efficient memory. J Immunol, 1995, 154(10): 5302-5312.
120. Szala S, Missol E, Sochanik A, et al. The use of cationic liposomes DC-CHOL/DOPE and DDAB/DOPE for direct transfer of Escherichia coli cytosine deaminase gene into growing melanoma tumors. Gene Ther, 1996, 3(11): 1026-1031.
121. Bruce JH, Harvey LN. Translating molecular imaging research into radiologic practice. Radiology, 2002, 222: 19-24.
122. O' Connell-Rodwell CE, Bums SM, Bachmann MH, et al. Bioluminescent indicators for in vivo measurements of gene expression. Trends in Biotechnology, 2002, 20(8Suppl.): S19-S23.
123. Freeman SM, Ramesh R, Marrogi AJ. Immune system in suicide-gene therapy. Lancet, 1997, 349(9044): 2-3.
124. Yamamoto S, Suzuki S, Hoshino A, et al. Herpes simplex virus thymidine kinase/ganciclovir-mediated killing of tumor cell induces tumor-specific cytotoxic T cells in mice. Cancer Gene Ther, 1997, 4(2): 91-96.
125. Haack K, Linnebacher M, Eisold S, et al. Induction of protective immunity against syngeneic rat cancer cells by expression of the cytosine deaminase suicide gene. Cancer Gene Ther, 2000, 7(10): 1357-1364.
126. Gavelli A, Baque P, Mala M, et al. Vaccination by suicide gene therapy against a model of hepatic metastasis from colon cancer in the rat. Ann Chir, 2000, 125(6): 552-559.
127. Nagy H J, Panis Y, Fabre M, et al. Efficient suicide gene therapy of transduced and distant untransduced ovary, tumors is correlated with significant increase of intratumoral T and NK cells. Biomed Pharmacother, 2000, 54(10): 479-486.
128.罗满林,顾为望主编.实验动物学.北京:中国农业出版社.2002,113-114.
129.Roda A, Pasini P, Mirasoli M, et al. Biotechnological applications of bioluminescence and chemiluminescence. Trends Biotechnol, 2004,22(6): 295-303.
130.Paroo Z, Bollinger RA, Braasch DA, et al. Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden. Mol Imaging, 2004,3(2): 117-124.
131.Shah K, Jacobs A, Breakefield XO, et al. Molecular imaging of gene therapy for cancer. Gene Ther. 2004, 11(15): 1175-1187.
132.Iyer M, Salazar FB, Lewis X, et al. Noninvasive imaging of enhanced prostate-specific gene expression using a two-step transcriptional amplification-based lentivirus vector. Mol Ther, 2004,10(3): 545-552.
133.Uhrbom L, Nerio E, Holland EC. Dissecting tumor maintenance requirements using bioluminescence imaging of cell proliferation in a mouse glioma model. Nat Med, 2004,10(11): 1257-1260.
134.Lyons SK. Advances in imaging mouse tumour models in vivo. J Pathol, 2005, 205(2): 194-205.