腺病毒介导的血管内皮生长因子启动子-胸苷激酶基因转移系统治疗肝癌的实验研究
|
摘要
{研究背景与目的}
肿瘤自杀基因疗法是目前认为最具有临床应用潜力的基因治疗策略之一，它
要求自杀基因能选择性地在靶肿瘤细胞中表达，而在正常细胞中不表达，以最大
限度地杀伤肿瘤细胞，最小限度地损伤正常组织。一个可能满足这一要求的方法
是利用肿瘤特异性基因调控元件驱动自杀基因表达。现已知道，肿瘤血管生成对
于肿瘤生长、浸润、转移都具有重要作用。这一复杂过程由肿瘤细胞分泌的血管
生成因子引发，其中血管内皮生长因子（vascular endothelial growth factor，
VEGF）起着关键作用。VEGF主要在肿瘤细胞以及受到肿瘤刺激的周围基质细胞
中表达。浸润性肿瘤常常血供不足，导致有些区域缺氧。无论是在体外或是在体
内，缺氧对VEGF基因表达均起着主要的调节作用。缺氧首先诱导产生缺氧诱导
因子HIF-1，后者与 VEGF 5‘侧区缺氧反应元件（HRE）结合，从而激活VEGF
基因表达。因此有可能以VEGF基因启动子设计肿瘤自杀基因疗法，利用肿瘤特
定的生存环境，如缺氧，来调控自杀基因选择性地在靶肿瘤细胞中表达，从而达
到治疗肿瘤的目的。
本研究将建立一种由腺病毒介导的、受VEGF启动子驱动的自杀基因转移系
统（AdVEGF-tk），利用该系统对体外培养的肝癌细胞和二乙基硝亚胺诱导的大鼠
肝癌模型进行治疗试验，期望VEGF启动子调控自杀基因HSV－tk在缺氧的肿瘤组
织中特异性表达，成为一个可能适用于多种恶性实体瘤的基因治疗方案。
｛方法｝
实验过程主要由三个部分组成：一采用简化的腺病毒载体构建系统AdEasy
System构建携带受人VEGF启动子调控HSV－tk基因表达的重组腺病毒载体
AdVEGF－tk。同时构建由巨细胞病毒（Cytomegaalovirus，CMV）强力启动子调控
TK基因表达的腺病毒载体AdCMV－TK，用作对照。
二 用重组腺病毒载体AdvEGF－tk和AdCMV－tk感染肝癌细胞株Hep-G2和
中文扩要
一
正常肝细胞L02这两种细胞，经过缺氧和不缺氧条件培养后，再予以GCV处理
5天后，用MTh法检测受染细胞对GCV的敏感性，以了解VEGF启动子能否调
控HSV－tk基因表达以及这种表达是否具有肿瘤细胞特异性和缺氧特异性。
三最后通过瘤内注射和肝动脉灌注法使重组腺病毒感染二乙基亚硝胺诱导
的WSfor大鼠肝癌，联合GCV处理，观察治疗前后肿瘤变化和大鼠生存率，以
了解 VEGF启动子能否调控 HSV－tk基因／GCV特异地杀伤体内肿瘤。
｛实验结果｝
通过AdEasy System我们构建成功高滴度的重组腺病毒载体AdVEGFrk和
AdCMV．h。在体外实验，携带受VEGF启动子调控HSV一比基因表达的重组
腺病毒载体 AdVEGF－h联合 GCV处理可杀伤在缺氧和不缺氧环境培养的肿瘤细
胞HepGZ而不杀伤在不缺氧环境培养的正常细胞L02，在缺氧条件下感染了
AdVEGF．tk的肿瘤细胞对GCV的敏感性明显要比不缺氧条件时高。在体内实验
中，瘤内注射腺病毒载体AdVEGF－tk，联合GCV处理可明显延缓大鼠肝脏肿瘤
生长速度；经肝动脉灌注AwEG卜比再加GCV处理，可明显减缓肿瘤生长并延
长大鼠的生存时间，也未观察到明显的毒副反应。
｛结论｝
实验结果表明，我们所构建的腺病毒介导的VEGF1K基因转移系统联合GCV
处理可特异地杀伤体内、体外缺氧的肿瘤细胞，且没有明显的毒副反应。由于
VEGF启动子具有大多数实体瘤所特有的缺氧反应活性，因此这一探索性结果有
可能应用于各种实体瘤的自杀基因治疗研究。
Most solid tumors have areas of low oxygen tension (hypoxia). Tumor cells under hypoxic conditions produce vascular endothelial growth factor (VEGF) to stimulate angiogenesis. The expression of the VEGF gene is regulated in hypoxic conditions by hypoxia-responsive elements (HREs), which are activated through the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Because normal tissues are not hypoxic, the presence of hypoxic cells provides the potential for designing cancer-specific gene therapy.
In the present study, we investigate whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the VEGF promoter encompassing the HRE site followed by GCV administration is effective in killing hepatocellular tumor cells in vitro and in experimental tumors.
We used the AdEasyTM system , which exploits E. coil’s robust, efficient recombination machinery instead of mammalian cells?to rapidly generate recombinant adenovirus without the need for time consuming plaque purification, to rapidly produce recombinant adenovirus AdVEGF-tk harboring the HSV-TK HSV-tk under the control of the human VEGF promoter 385bp sequence (from -1175bp to -790bp) encompassing the hypoxia response element (HRE). Then, recombinant adenovirus AdVEGF-tk was transduced into hepatoma cell line Hep-G2 and normal liver cell line L02 under both oxic and anoxic conditions followed by GCV administration for 5 days. Cell survival assays demonstrated GCV killing of Hep-G2 cells under both oxic and anoxic conditions, but not of L02 cells under oxic condition. Moreover, Hep-G2 cells infected with AdVEGF-tk showed the increased GCV sensitivity under anoxic
as compared with oxic conditions. In vivo, the evaluation of the efficiency of Ad.VEGGF-tk /GCV on tumor growth was assessed in a relevant model of multifocal hepatic lesions induced in rats by a potent alkylating chemical carcinogen, diethylnitrosamine using intratumoral or intrahepatic artery injection of the recombinant AdVEGF-tk. The results displayed a pronounced tumors growth delay and a prolonged survival time when Ad.VEGF-tk was injected by intratumoral (lxi O9pfu) or intrahepatic (5x1 O9pfu) artery route followed by GCV administrations. Further, no significant adverse toxicity was shown in the rats treated with AdVEGF-tk plus GCV by the intrahepatic artery route.
These results indicate that the hypoxia-inducible promoter of the human vascular endothelial growth factor gene could increase the selective tumoricidal activity by GCV in gene therapy for hepatocellular carcinoma. Because hypoxia occurs in many solid tumors, this approach would be applicable to a wide range of solid tumors.
肿瘤自杀基因疗法是目前认为最具有临床应用潜力的基因治疗策略之一，它
要求自杀基因能选择性地在靶肿瘤细胞中表达，而在正常细胞中不表达，以最大
限度地杀伤肿瘤细胞，最小限度地损伤正常组织。一个可能满足这一要求的方法
是利用肿瘤特异性基因调控元件驱动自杀基因表达。现已知道，肿瘤血管生成对
于肿瘤生长、浸润、转移都具有重要作用。这一复杂过程由肿瘤细胞分泌的血管
生成因子引发，其中血管内皮生长因子（vascular endothelial growth factor，
VEGF）起着关键作用。VEGF主要在肿瘤细胞以及受到肿瘤刺激的周围基质细胞
中表达。浸润性肿瘤常常血供不足，导致有些区域缺氧。无论是在体外或是在体
内，缺氧对VEGF基因表达均起着主要的调节作用。缺氧首先诱导产生缺氧诱导
因子HIF-1，后者与 VEGF 5‘侧区缺氧反应元件（HRE）结合，从而激活VEGF
基因表达。因此有可能以VEGF基因启动子设计肿瘤自杀基因疗法，利用肿瘤特
定的生存环境，如缺氧，来调控自杀基因选择性地在靶肿瘤细胞中表达，从而达
到治疗肿瘤的目的。
本研究将建立一种由腺病毒介导的、受VEGF启动子驱动的自杀基因转移系
统（AdVEGF-tk），利用该系统对体外培养的肝癌细胞和二乙基硝亚胺诱导的大鼠
肝癌模型进行治疗试验，期望VEGF启动子调控自杀基因HSV－tk在缺氧的肿瘤组
织中特异性表达，成为一个可能适用于多种恶性实体瘤的基因治疗方案。
｛方法｝
实验过程主要由三个部分组成：一采用简化的腺病毒载体构建系统AdEasy
System构建携带受人VEGF启动子调控HSV－tk基因表达的重组腺病毒载体
AdVEGF－tk。同时构建由巨细胞病毒（Cytomegaalovirus，CMV）强力启动子调控
TK基因表达的腺病毒载体AdCMV－TK，用作对照。
二 用重组腺病毒载体AdvEGF－tk和AdCMV－tk感染肝癌细胞株Hep-G2和
中文扩要
一
正常肝细胞L02这两种细胞，经过缺氧和不缺氧条件培养后，再予以GCV处理
5天后，用MTh法检测受染细胞对GCV的敏感性，以了解VEGF启动子能否调
控HSV－tk基因表达以及这种表达是否具有肿瘤细胞特异性和缺氧特异性。
三最后通过瘤内注射和肝动脉灌注法使重组腺病毒感染二乙基亚硝胺诱导
的WSfor大鼠肝癌，联合GCV处理，观察治疗前后肿瘤变化和大鼠生存率，以
了解 VEGF启动子能否调控 HSV－tk基因／GCV特异地杀伤体内肿瘤。
｛实验结果｝
通过AdEasy System我们构建成功高滴度的重组腺病毒载体AdVEGFrk和
AdCMV．h。在体外实验，携带受VEGF启动子调控HSV一比基因表达的重组
腺病毒载体 AdVEGF－h联合 GCV处理可杀伤在缺氧和不缺氧环境培养的肿瘤细
胞HepGZ而不杀伤在不缺氧环境培养的正常细胞L02，在缺氧条件下感染了
AdVEGF．tk的肿瘤细胞对GCV的敏感性明显要比不缺氧条件时高。在体内实验
中，瘤内注射腺病毒载体AdVEGF－tk，联合GCV处理可明显延缓大鼠肝脏肿瘤
生长速度；经肝动脉灌注AwEG卜比再加GCV处理，可明显减缓肿瘤生长并延
长大鼠的生存时间，也未观察到明显的毒副反应。
｛结论｝
实验结果表明，我们所构建的腺病毒介导的VEGF1K基因转移系统联合GCV
处理可特异地杀伤体内、体外缺氧的肿瘤细胞，且没有明显的毒副反应。由于
VEGF启动子具有大多数实体瘤所特有的缺氧反应活性，因此这一探索性结果有
可能应用于各种实体瘤的自杀基因治疗研究。
Most solid tumors have areas of low oxygen tension (hypoxia). Tumor cells under hypoxic conditions produce vascular endothelial growth factor (VEGF) to stimulate angiogenesis. The expression of the VEGF gene is regulated in hypoxic conditions by hypoxia-responsive elements (HREs), which are activated through the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Because normal tissues are not hypoxic, the presence of hypoxic cells provides the potential for designing cancer-specific gene therapy.
In the present study, we investigate whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the VEGF promoter encompassing the HRE site followed by GCV administration is effective in killing hepatocellular tumor cells in vitro and in experimental tumors.
We used the AdEasyTM system , which exploits E. coil’s robust, efficient recombination machinery instead of mammalian cells?to rapidly generate recombinant adenovirus without the need for time consuming plaque purification, to rapidly produce recombinant adenovirus AdVEGF-tk harboring the HSV-TK HSV-tk under the control of the human VEGF promoter 385bp sequence (from -1175bp to -790bp) encompassing the hypoxia response element (HRE). Then, recombinant adenovirus AdVEGF-tk was transduced into hepatoma cell line Hep-G2 and normal liver cell line L02 under both oxic and anoxic conditions followed by GCV administration for 5 days. Cell survival assays demonstrated GCV killing of Hep-G2 cells under both oxic and anoxic conditions, but not of L02 cells under oxic condition. Moreover, Hep-G2 cells infected with AdVEGF-tk showed the increased GCV sensitivity under anoxic
as compared with oxic conditions. In vivo, the evaluation of the efficiency of Ad.VEGGF-tk /GCV on tumor growth was assessed in a relevant model of multifocal hepatic lesions induced in rats by a potent alkylating chemical carcinogen, diethylnitrosamine using intratumoral or intrahepatic artery injection of the recombinant AdVEGF-tk. The results displayed a pronounced tumors growth delay and a prolonged survival time when Ad.VEGF-tk was injected by intratumoral (lxi O9pfu) or intrahepatic (5x1 O9pfu) artery route followed by GCV administrations. Further, no significant adverse toxicity was shown in the rats treated with AdVEGF-tk plus GCV by the intrahepatic artery route.
These results indicate that the hypoxia-inducible promoter of the human vascular endothelial growth factor gene could increase the selective tumoricidal activity by GCV in gene therapy for hepatocellular carcinoma. Because hypoxia occurs in many solid tumors, this approach would be applicable to a wide range of solid tumors.
引文
1． GRECO O AND DACHS GU. Gene Directed Enzyme/Prodrug Therapy of Cancer: Historical Appraisal and Future Prospectives. J. Cell. Physi. 2001． 187:22-36．
2． Springer C J and Niculescu-Duvaz I. Prodrug-activating systems in suicide gene therapy. J. Clinical Investigation. 2000． 105(9) : 1161-1167．
3． Fumihiko K, Hirofumi H, Yasushi S, et al. Adenovirus-mediated enzyme-prodrug therapy for cancer. Cancer Journal. 1997． 10 (6) : 353-48．
4． Dachs GU, Dougherty GJ, Stratford IJ, et al. Targeting gene therapy to cancer: a review. Oncol Res 1997; 9 (6-7) : 313-25．
5． Kanai F, Lan KH, Shiratori T. In vitro gene therapy for fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosinedeaminase gene. Cancer Res. 1997． 57:461-474．
6． Lechanteur C, Princer F, Lo-Bue S, et al. HSV-1 thymidine kinase gene therapy for colorectal adenocarcinoma-derived peritoneal carcinomatosis. Gene Ther. 1997． 4:1189-1194．
7． Qian C, Idonte M, Bilbao R. Gene transfer and therapy with adenoviral rector in rats with diethylnitrosamine-induced hepatocellular carcinoma. Hum Gene Ther. 1997． 8:349-358．
8． Huber BE, Richards CA, Krenitsky TA. Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma an innovative approach for cancer therapy. Proc Natl Acad Sci USA. 1991． 88:8039-8093．
9． Brand K, Arnold W, Bartels T. Liver-associated toxicity of the HSV-tk/GCV approach and adenoviral vectors. Cancer Gene Ther. 1997． 4:9-16．
10． Grunstein J, Roberts WG, Mathieu-Costello O, et al. Tumor-derived Expression of Vascular Endothelial Growth Factor Is a Critical Factor in Tumor Expansion and Vascular Function. Cancer Res.1999． 59: 1592-1598．
11． Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature (Lond.). 1992． 359: 843-845．
12． Plate K. H., Breier G., Weich H. A., Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature (Lond.). 1992． 359:845-848,
13． Forsythe JA, Jiang B-H, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor-1． Mol. Cell. Biol. 1996． 16:4604-4613．
14． Mise M., Arii S., Higashitsuji H., Furutani M., Niwano M., Harada T., Ishigami S., Toda Y, Nakayama H., Fukumoto M., Fujita J., Imamura M. Clinical significance of vascular endothelial growth factor and basic fibroblast growth factor gene expression in liver tumor. Hepatology. 1996． 23:455-464．
15． Suzuki K, Hayashi N, Miyamoto Y, et al. Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma. Cancer Res. 1996． 56:3004-3009．
16． Yamaguchi R, Yano H, lemura A, et al. Expression of vascular endothelial growth factor in human hepatocellular carcinoma. Hepatology. 1998． 28: 68-77．
17． Tamaoki T., Fausto N. Expression of a-fetoprotein gene during development, regeneration, and carcinogenesis Stein G. Stein J. eds. Recombinant DNA and Cell Proliferation: 145-168, Academic Press Orlando, FL 1984．
18． Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter. Cancer Res. 2001． 1; 61 (7) : 3016-21．
19． Mazure NM, Chen EY, Yeh P, et al. Oncogenic transformation and hypoxia synergistically act to modulate vascular endothelial growth factor expression. Cancer Res. 1996． 56(15) : 3436-40．
20． Kuriyama S, Mitoro A, Yamazaki M, et al. Comparison of gene therapy with the herpes simplex virus thymidine kinase gene and the bacterial cytosine deaminase gene for the treatment of hepatocellular carcinoma. Scand.J..Gastroenterol. 1999． 34 (10) : 1033-41．
21． HE TC, ZHOU S, LUIS T, et al. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA. 1998． 95: 2509-2514．
22． Kuriyama S. Cancer gene therapy with HSV-tk/GCV system depends on T-cell-mediated immune responses and causes apoptotic death of tumor cells in vivo. Int J Cancer. 1999． 83(3) : 374-80．
23． Gerolami R, Cardoso J, Bralet MP, et al. Enhanced in vivo adenovirus-mediated gene transfer to rat hepatocarcinomas by selective administration into the hepatic artery. Gene Ther. 1998． 5: 896-904．
24． Thomas KR and Capecchi M R. Site-directed mutagenesis by gene targeting in mousseembryo-derived stem cells. Cell.1987． 51: 503-512．
25． Mansour SL, Thomas KR, Capecchi MR. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988． 336:348-352．
26． J 萨姆布鲁克,EF弗里奇,T曼尼阿蒂斯著。1992。金冬雁,黎孟枫等译。分子克隆实验指南。第二版。北京:科学出版社
27． Koji K, Koji N, Hiroaki N. Plasm id-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sabcBC mutant involvement of RecF recombination pathway genes. J.Mol.Biol.1989． 209: 623-634．
28． Kaneko S, Hallenbeck P, Kotani T, et al. Adenovirus-mediated gene therapy of hepatocellular carcinoma using cancer-specific gene expression. Cancer Res 1995． 55(22) : 5283-7．
29． Shibata T, Akiyama N, Noda M, et al. Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes. Int J Radiat Oncol Biol Phys. 1998 .42(4) : 913-6．
30． Gerolami R, Cardoso J, Lewin M, et al. Evaluation of HSV-tk gene therapy in a rat model of chemically induced hepatocellular carcinoma by intratumoral and intrahepatic artery routes. Cancer Res. 2000． 60 (4) : 993-1001．
31． Barajas M, Mazzolini G, Genove G, et al. Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12． Hepatology. 2001． 33(1) : 52-61．
32． Rajewsky M F. Changes in DMA synthesis and cell proliferation during hepatocarcinogenesis by diethylnitrosamine. Eur. J. Cancer. 1967． 3: 335-342,
33． Rabes H. M., Szymkowiak R. Cell kinetics of hepatocytes during the
preneoplastic period of diethylnitrosamine-induced liver carcinogenesis. Cancer Res. 1979． 39:1298-1304．
34． Gervaz P, Scholl B, Padrum V, et al. Growth inhibition of liver metastases by the anti-angiogenic drug TNP-470． Liver. 2000． 20: 108-113．
35． Fukumura D, Xavier R, Sugiura T, et al. Tumor induction of VEGF promoter activity in stromal cells. Cell. 1998． 94:715-725．
36． Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1． Mol Cell Biol. 1996 .16(9) : 4604-13
37． Kianmanesh AR, Perrin H, Panis Y, et al. A "distant" bystander effect of suicide gene therapy: regression of nontransduced tumors together with a distant transduced tumor. Hum. Gene Ther. 1997． 8:1807-1814．
38． Bramson JL, Hitt M, Gauldie J, et al. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination. Gene Then 1997． 4:1069-1076．
39． Dachs GU, Patterson AV, Firth JD, et al. Targeting gene expression to hypoxic tumor cells. Nat. Med. 1997． 3:515-520．
2． Springer C J and Niculescu-Duvaz I. Prodrug-activating systems in suicide gene therapy. J. Clinical Investigation. 2000． 105(9) : 1161-1167．
3． Fumihiko K, Hirofumi H, Yasushi S, et al. Adenovirus-mediated enzyme-prodrug therapy for cancer. Cancer Journal. 1997． 10 (6) : 353-48．
4． Dachs GU, Dougherty GJ, Stratford IJ, et al. Targeting gene therapy to cancer: a review. Oncol Res 1997; 9 (6-7) : 313-25．
5． Kanai F, Lan KH, Shiratori T. In vitro gene therapy for fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosinedeaminase gene. Cancer Res. 1997． 57:461-474．
6． Lechanteur C, Princer F, Lo-Bue S, et al. HSV-1 thymidine kinase gene therapy for colorectal adenocarcinoma-derived peritoneal carcinomatosis. Gene Ther. 1997． 4:1189-1194．
7． Qian C, Idonte M, Bilbao R. Gene transfer and therapy with adenoviral rector in rats with diethylnitrosamine-induced hepatocellular carcinoma. Hum Gene Ther. 1997． 8:349-358．
8． Huber BE, Richards CA, Krenitsky TA. Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma an innovative approach for cancer therapy. Proc Natl Acad Sci USA. 1991． 88:8039-8093．
9． Brand K, Arnold W, Bartels T. Liver-associated toxicity of the HSV-tk/GCV approach and adenoviral vectors. Cancer Gene Ther. 1997． 4:9-16．
10． Grunstein J, Roberts WG, Mathieu-Costello O, et al. Tumor-derived Expression of Vascular Endothelial Growth Factor Is a Critical Factor in Tumor Expansion and Vascular Function. Cancer Res.1999． 59: 1592-1598．
11． Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature (Lond.). 1992． 359: 843-845．
12． Plate K. H., Breier G., Weich H. A., Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature (Lond.). 1992． 359:845-848,
13． Forsythe JA, Jiang B-H, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor-1． Mol. Cell. Biol. 1996． 16:4604-4613．
14． Mise M., Arii S., Higashitsuji H., Furutani M., Niwano M., Harada T., Ishigami S., Toda Y, Nakayama H., Fukumoto M., Fujita J., Imamura M. Clinical significance of vascular endothelial growth factor and basic fibroblast growth factor gene expression in liver tumor. Hepatology. 1996． 23:455-464．
15． Suzuki K, Hayashi N, Miyamoto Y, et al. Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma. Cancer Res. 1996． 56:3004-3009．
16． Yamaguchi R, Yano H, lemura A, et al. Expression of vascular endothelial growth factor in human hepatocellular carcinoma. Hepatology. 1998． 28: 68-77．
17． Tamaoki T., Fausto N. Expression of a-fetoprotein gene during development, regeneration, and carcinogenesis Stein G. Stein J. eds. Recombinant DNA and Cell Proliferation: 145-168, Academic Press Orlando, FL 1984．
18． Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter. Cancer Res. 2001． 1; 61 (7) : 3016-21．
19． Mazure NM, Chen EY, Yeh P, et al. Oncogenic transformation and hypoxia synergistically act to modulate vascular endothelial growth factor expression. Cancer Res. 1996． 56(15) : 3436-40．
20． Kuriyama S, Mitoro A, Yamazaki M, et al. Comparison of gene therapy with the herpes simplex virus thymidine kinase gene and the bacterial cytosine deaminase gene for the treatment of hepatocellular carcinoma. Scand.J..Gastroenterol. 1999． 34 (10) : 1033-41．
21． HE TC, ZHOU S, LUIS T, et al. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA. 1998． 95: 2509-2514．
22． Kuriyama S. Cancer gene therapy with HSV-tk/GCV system depends on T-cell-mediated immune responses and causes apoptotic death of tumor cells in vivo. Int J Cancer. 1999． 83(3) : 374-80．
23． Gerolami R, Cardoso J, Bralet MP, et al. Enhanced in vivo adenovirus-mediated gene transfer to rat hepatocarcinomas by selective administration into the hepatic artery. Gene Ther. 1998． 5: 896-904．
24． Thomas KR and Capecchi M R. Site-directed mutagenesis by gene targeting in mousseembryo-derived stem cells. Cell.1987． 51: 503-512．
25． Mansour SL, Thomas KR, Capecchi MR. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988． 336:348-352．
26． J 萨姆布鲁克,EF弗里奇,T曼尼阿蒂斯著。1992。金冬雁,黎孟枫等译。分子克隆实验指南。第二版。北京:科学出版社
27． Koji K, Koji N, Hiroaki N. Plasm id-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sabcBC mutant involvement of RecF recombination pathway genes. J.Mol.Biol.1989． 209: 623-634．
28． Kaneko S, Hallenbeck P, Kotani T, et al. Adenovirus-mediated gene therapy of hepatocellular carcinoma using cancer-specific gene expression. Cancer Res 1995． 55(22) : 5283-7．
29． Shibata T, Akiyama N, Noda M, et al. Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes. Int J Radiat Oncol Biol Phys. 1998 .42(4) : 913-6．
30． Gerolami R, Cardoso J, Lewin M, et al. Evaluation of HSV-tk gene therapy in a rat model of chemically induced hepatocellular carcinoma by intratumoral and intrahepatic artery routes. Cancer Res. 2000． 60 (4) : 993-1001．
31． Barajas M, Mazzolini G, Genove G, et al. Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12． Hepatology. 2001． 33(1) : 52-61．
32． Rajewsky M F. Changes in DMA synthesis and cell proliferation during hepatocarcinogenesis by diethylnitrosamine. Eur. J. Cancer. 1967． 3: 335-342,
33． Rabes H. M., Szymkowiak R. Cell kinetics of hepatocytes during the
preneoplastic period of diethylnitrosamine-induced liver carcinogenesis. Cancer Res. 1979． 39:1298-1304．
34． Gervaz P, Scholl B, Padrum V, et al. Growth inhibition of liver metastases by the anti-angiogenic drug TNP-470． Liver. 2000． 20: 108-113．
35． Fukumura D, Xavier R, Sugiura T, et al. Tumor induction of VEGF promoter activity in stromal cells. Cell. 1998． 94:715-725．
36． Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1． Mol Cell Biol. 1996 .16(9) : 4604-13
37． Kianmanesh AR, Perrin H, Panis Y, et al. A "distant" bystander effect of suicide gene therapy: regression of nontransduced tumors together with a distant transduced tumor. Hum. Gene Ther. 1997． 8:1807-1814．
38． Bramson JL, Hitt M, Gauldie J, et al. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination. Gene Then 1997． 4:1069-1076．
39． Dachs GU, Patterson AV, Firth JD, et al. Targeting gene expression to hypoxic tumor cells. Nat. Med. 1997． 3:515-520．
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |