HBV启动子调控的肝细胞高表达载体构建及抑癌作用研究
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摘要
肝癌是最常见的恶性肿瘤之一，每年使大约100万例病人死亡，尽管目前肝癌的治疗方法很多，但仍未能从根本上改变病人的预后。近年来肝癌的基因治疗已经成为生物医学和临床医学研究的热点。肝癌基因治疗的靶向性调控，即调控目的基因特异性导入肝癌细胞或在肝癌细胞中实现特异性表达，最大限度的杀伤肝癌细胞的同时而不损伤正常肝细胞，已成为决定基因治疗效果及可行性的关键因素。
分子病毒学研究揭示了HBV的表达调控机制，为利用HBV嗜肝细胞基因表达调控特性构建肝细胞特异表达载体提供了条件。HBV基因组仅有3.2kb，结构紧密，所有调控序列均位于蛋白编码区。HBV 3.5kb、2.4kb、2.1kb和0.9kb的RNA转录物的起始点附近各有一个启动子，分别为C、SPⅠ、SPⅡ和X启动子。另外在1074～1234位核苷酸和1627～1774位核苷酸处有HBV的两个增强子ENⅠ和ENⅡ。该两段序列具有高度保守性，并在其上有多个肝特异转录因子的结合位点，与ENⅠ结合的转录因子有肝细胞特异性的C／EBP、HNF-4和HB_1F，与ENⅡ相结合的肝细胞蛋白因子有C／EBP、HNF-4、HLF、HNF3、FTF和E4BP4等。由于与HBV的启动子和增强子特异结合的转录表达调控因子几乎只存在于肝细胞中，而不存在于其它细胞中，决定了肝细胞增强子ENⅠ和ENⅡ具有高度肝细胞专一性，其对HBV在肝细胞中的特异表达有相当重要的贡献，并在一定程度上决定了HBV的嗜肝性。
因此，以HBV病毒嗜肝细胞表达密切相关的启动子、增强子为表达调控元件构建肝细胞特异表达载体携带只对肿瘤细胞起抑制或促调亡作用的治疗基因导入人体后，便可达到杀伤肝癌细胞而对正常肝细胞或非肝细胞无影响的目的，充分体现了基因治疗的优势。据此，本论文拟进行以下研究：(1) 克隆不同的HBV启动子片段，评价其在肝细胞和非肝细胞中的转录活性。筛选出在肝细胞中特异性表达最强的DNA序列组合形式。(2) 构建HBV启动子调控的外源端粒酶hTRT反义RNA、apoptin、IL-24和OSM基因载体，并检测各种治疗基因在体内外的抑癌作用。
硕士学位论文
中文摘要
主要工作及结果概述如下
(一)肝细胞特异表达载体的构建
1.构建了受HBV EN 11加基本核心启动子(BCP)、ENI和EN 11加BCP联合调控、以
及ENH加BCP联合mCMV调控的五种荧光素酶报告载体。
2.筛选获得了肝细胞高效特异表达的载体
应用脂质体介导基因转染技术将此5种质粒转染HePGZ、22.15、BEL一7402、
SNI入IC7721等4种肝癌细胞和肝细胞LOZ以及5种非肝细胞Hela、446、MCF7、803、
A375，双荧光素酶报告基因实验检测其活性。pEN nR(突变型)在各种肝细胞中的转
录活性为阳性对照sV40启动子的0.98界5.79倍，pENI一HW(a丫囚型)为在肝细胞中转
录活性为阳性对照SV40启动子的1 .25一巧.31倍，pENI一nR(突变型)在肝细胞转录活性
为阳性对照SV4O启动子的3 .76~21 .83倍，pEN H mCMV在肝细胞转录活性可为阳性
对照SV40启动子的9.4弘91 .71倍;同时发玫哩于生型EN HW启动子在5种肝细胞中的
转录活性分别为SV4O启动子转录活性的1 .32一11 .12倍，而在5种非肝细胞中转录活性
仅为SV40启动子的2935%司27%，提示HBV野生型EN nw启动子具有明显的嗜肝
特异性表达的特性，该结果为特异性抗HBV及抗肝癌药物评价系统的建立创造了条件。
3.获得了一条新型HBV启动子序列
以漫性HBV病人的血清为模板，PCR扩增出HBV基因组中含ENI和ENn及核心
启动子(BCP)序列的片段，Cp区序列存在变异，但cp区突变并没有降低该启动子
转录活性，该HBV启动子在肝细胞中均具有较强的转录活性，尤其是在稳定转染了乙
肝病毒的2.2.15细胞中转录活性更高。该突变序列在GeneBank中未注册过，为本实验
首次发现新序列。
(二)HBv启动子调控表达的外源端粒酶hTRT反义RNA、aP叩tin、IL一4和OSM
基因载体的构建
构建了肝细胞高表达HBv启动子及SV40启动子调控的apoptin基因及反义hl，Rl，
的真核表达载体pEN 11 W-aPo、psV4o一即。、p刚11 w-ashTRT(1.2比)、
PSV40一ash耳汀(l .2kb)，另外还构建了肝细胞高表达HBv启动子及SV40启动子调控的
OSM基因、几一24基因的真核表达载体p刚IIW‘osm、pEN 11 W-24、pSV40一24。将以上
获得的各载体转染肝癌细胞HePGZ，通过RT-PcR分别检测HBV启动子和SV40启动子
硕士学位论文
中文摘要
驱动鲜力ptin、IL一24、反义hTm，和osm基因在HePGZ细胞中的转录情况，结果表明在
肝癌细胞中均可检测到外源即叩tin、IL一24、反义h们砚，基因和osm基因的转录。
(三)HBv启动子调控表达的端粒酶hTRT反义RNA、aP叩tin、IL24和osM基因
载体体内外抑癌作用
1.体外抑癌作用
为了研究aP0ptin、IL.24、反义hl，Rl，和osm基因表达对肝癌细胞和非肝癌细胞的作
用，采用MTT法和流式细胞法分别检测它们对肝癌细胞和非肝癌细胞增殖抑制的影响
和诱导细胞凋亡的情况。结果显示以上四种载体在三种肝癌细胞HepGZ、7402、7721
中均出现明显的细胞增殖抑制现象， pEN H W-24转染抑制率为11.6%卜37.6%，pENH
W-aPoPtin为20.0%一33.7%，pEN IIW-osm为13.8叹产29.9%，pENllw-ashTRT为12.2%
砚1 .9%。pEN H w-osm在肝癌细胞中可引起一定程度
Hepatocellular carcinoma(HCC) is one of the most common maliganant tumors in the world, responsible for an estimated one million deaths annually. The prognosis of HCC patients is generally very poor despite many treatment strategies. Gene therapy is an exciting approach to treat HCC in the biological and clinical research. The success of gene therapy largely depends on the development of a vector or vehicle that can selectively and efficiently deliver a gene to tumor cells or express therapeutic gene only in tumor tissues with minimal toxicity in normal hepatic cells or nonhepatic cells. Study of the molecular virology revealed the mechanism of the expression and regulation of HBV, which may attribute to the construction of hepatic-specific expression system of vector. HBV has a partially double-stranded 3.2-kb DNA genome, and all the regulating sequence located in the protein encoding region. Four promoters(Cp, SP I, SPII, Xp) have been identified as cis regulators for transcription of the 3.5-, 2.4-, 2.1-a
nd 0.8-kb mRNAs, respectively. Two regions(nt1074 to 1234, nt1627 to 1774) in the HBV genome have been shown to act as transcriptional enhancers, Enhancer I (EN I )and Enhancer II(EN II), which show highly conservative trait, A lot of hepatocyte-specific factors bind to those two regions. For instance, C/EBP, HNF-4 and HB1F bind to EN I region, and the transcriptional factors, like C/EBP, HNF-4, HLF, HNF3, FTF and E4BP4, bind to EN II region. Only in the hepatocyte cells can these transcriptional factors be detected, but not in nonhepatic cells. This suggests that these transcriptional factors are necessary for the hepatocyte-specific traits of the EN I and EN II, and the hepatotropism of HBV replication might be related with these transcriptional factors.
Therefore we use the transcriptional regulatory sequence which might be attributable to the hepatotropism of HBV and use the therapic genes which only inhibit or kill tumor cells to construct the hepatic gene therapy vectors. When transferred into body, they will kill the hepatic tumor cells without damaging the normal cells. So we carry out our studies as follows: (1) Cloning of different HBV promoters and assessing of transcriptional activity of HBV promoter in hepatocyteor nonhepatic cells. (2) Construction of apoptin, antisense hTRT, IL-24 and
oncostatin M(osm) eukaryotic expression vectors driven by the HBV promoter and study of its expression specificity and anticancer effect in vitro and in vivo.
The preliminary research results as follows:
I. Construction of hepatocyte-specific vectors
1. Five Luciferase reporter plasmids driven by HBV EN II with basic core promoter(BCP), EN I and EN II with BCP, HBV EN II and BCP with mCMV promoter were successfully constructed.
2. A high hepatocyte-specific expression vector was achieved
The recombinant plasmids were transfercted into five hepatic cells(HepG2, 2.2.15, BEL-7402, SMMC7721, LO2) and five nonhepatic cells(HeLa, H460, MCF7, 803, A375)using Lipofectamine?reagent, The Luciferase expression which indirectly represented the transcriptional activeity of HBV promoter lying upstream of Luc gene was detected with Dual-Luciferase Reporter Assay System. the SV40 enhancer/promoter (pGL3-Control) as a positive control, and the negative control without promoter (pGL3-Basic). The Luciferase activity of pGL3-Control plasmid in each cell line was considered as 100%. The HBV promoter construct showed significant transcriptional activity in all hepatic cell lines., the transcriptional activity of EN IIR (mutant type) promoter was 0.987-5.79 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of EN I -IlW(ayw type) promoter was 1.25-15.31 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of EN I -IIR promoter was 3.76-21.83 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of ENIImCMV promoter was 9.49-91.71 fold of positive control under SV40 promoter in hepatic cell lines
分子病毒学研究揭示了HBV的表达调控机制，为利用HBV嗜肝细胞基因表达调控特性构建肝细胞特异表达载体提供了条件。HBV基因组仅有3.2kb，结构紧密，所有调控序列均位于蛋白编码区。HBV 3.5kb、2.4kb、2.1kb和0.9kb的RNA转录物的起始点附近各有一个启动子，分别为C、SPⅠ、SPⅡ和X启动子。另外在1074～1234位核苷酸和1627～1774位核苷酸处有HBV的两个增强子ENⅠ和ENⅡ。该两段序列具有高度保守性，并在其上有多个肝特异转录因子的结合位点，与ENⅠ结合的转录因子有肝细胞特异性的C／EBP、HNF-4和HB_1F，与ENⅡ相结合的肝细胞蛋白因子有C／EBP、HNF-4、HLF、HNF3、FTF和E4BP4等。由于与HBV的启动子和增强子特异结合的转录表达调控因子几乎只存在于肝细胞中，而不存在于其它细胞中，决定了肝细胞增强子ENⅠ和ENⅡ具有高度肝细胞专一性，其对HBV在肝细胞中的特异表达有相当重要的贡献，并在一定程度上决定了HBV的嗜肝性。
因此，以HBV病毒嗜肝细胞表达密切相关的启动子、增强子为表达调控元件构建肝细胞特异表达载体携带只对肿瘤细胞起抑制或促调亡作用的治疗基因导入人体后，便可达到杀伤肝癌细胞而对正常肝细胞或非肝细胞无影响的目的，充分体现了基因治疗的优势。据此，本论文拟进行以下研究：(1) 克隆不同的HBV启动子片段，评价其在肝细胞和非肝细胞中的转录活性。筛选出在肝细胞中特异性表达最强的DNA序列组合形式。(2) 构建HBV启动子调控的外源端粒酶hTRT反义RNA、apoptin、IL-24和OSM基因载体，并检测各种治疗基因在体内外的抑癌作用。
硕士学位论文
中文摘要
主要工作及结果概述如下
(一)肝细胞特异表达载体的构建
1.构建了受HBV EN 11加基本核心启动子(BCP)、ENI和EN 11加BCP联合调控、以
及ENH加BCP联合mCMV调控的五种荧光素酶报告载体。
2.筛选获得了肝细胞高效特异表达的载体
应用脂质体介导基因转染技术将此5种质粒转染HePGZ、22.15、BEL一7402、
SNI入IC7721等4种肝癌细胞和肝细胞LOZ以及5种非肝细胞Hela、446、MCF7、803、
A375，双荧光素酶报告基因实验检测其活性。pEN nR(突变型)在各种肝细胞中的转
录活性为阳性对照sV40启动子的0.98界5.79倍，pENI一HW(a丫囚型)为在肝细胞中转
录活性为阳性对照SV40启动子的1 .25一巧.31倍，pENI一nR(突变型)在肝细胞转录活性
为阳性对照SV4O启动子的3 .76~21 .83倍，pEN H mCMV在肝细胞转录活性可为阳性
对照SV40启动子的9.4弘91 .71倍;同时发玫哩于生型EN HW启动子在5种肝细胞中的
转录活性分别为SV4O启动子转录活性的1 .32一11 .12倍，而在5种非肝细胞中转录活性
仅为SV40启动子的2935%司27%，提示HBV野生型EN nw启动子具有明显的嗜肝
特异性表达的特性，该结果为特异性抗HBV及抗肝癌药物评价系统的建立创造了条件。
3.获得了一条新型HBV启动子序列
以漫性HBV病人的血清为模板，PCR扩增出HBV基因组中含ENI和ENn及核心
启动子(BCP)序列的片段，Cp区序列存在变异，但cp区突变并没有降低该启动子
转录活性，该HBV启动子在肝细胞中均具有较强的转录活性，尤其是在稳定转染了乙
肝病毒的2.2.15细胞中转录活性更高。该突变序列在GeneBank中未注册过，为本实验
首次发现新序列。
(二)HBv启动子调控表达的外源端粒酶hTRT反义RNA、aP叩tin、IL一4和OSM
基因载体的构建
构建了肝细胞高表达HBv启动子及SV40启动子调控的apoptin基因及反义hl，Rl，
的真核表达载体pEN 11 W-aPo、psV4o一即。、p刚11 w-ashTRT(1.2比)、
PSV40一ash耳汀(l .2kb)，另外还构建了肝细胞高表达HBv启动子及SV40启动子调控的
OSM基因、几一24基因的真核表达载体p刚IIW‘osm、pEN 11 W-24、pSV40一24。将以上
获得的各载体转染肝癌细胞HePGZ，通过RT-PcR分别检测HBV启动子和SV40启动子
硕士学位论文
中文摘要
驱动鲜力ptin、IL一24、反义hTm，和osm基因在HePGZ细胞中的转录情况，结果表明在
肝癌细胞中均可检测到外源即叩tin、IL一24、反义h们砚，基因和osm基因的转录。
(三)HBv启动子调控表达的端粒酶hTRT反义RNA、aP叩tin、IL24和osM基因
载体体内外抑癌作用
1.体外抑癌作用
为了研究aP0ptin、IL.24、反义hl，Rl，和osm基因表达对肝癌细胞和非肝癌细胞的作
用，采用MTT法和流式细胞法分别检测它们对肝癌细胞和非肝癌细胞增殖抑制的影响
和诱导细胞凋亡的情况。结果显示以上四种载体在三种肝癌细胞HepGZ、7402、7721
中均出现明显的细胞增殖抑制现象， pEN H W-24转染抑制率为11.6%卜37.6%，pENH
W-aPoPtin为20.0%一33.7%，pEN IIW-osm为13.8叹产29.9%，pENllw-ashTRT为12.2%
砚1 .9%。pEN H w-osm在肝癌细胞中可引起一定程度
Hepatocellular carcinoma(HCC) is one of the most common maliganant tumors in the world, responsible for an estimated one million deaths annually. The prognosis of HCC patients is generally very poor despite many treatment strategies. Gene therapy is an exciting approach to treat HCC in the biological and clinical research. The success of gene therapy largely depends on the development of a vector or vehicle that can selectively and efficiently deliver a gene to tumor cells or express therapeutic gene only in tumor tissues with minimal toxicity in normal hepatic cells or nonhepatic cells. Study of the molecular virology revealed the mechanism of the expression and regulation of HBV, which may attribute to the construction of hepatic-specific expression system of vector. HBV has a partially double-stranded 3.2-kb DNA genome, and all the regulating sequence located in the protein encoding region. Four promoters(Cp, SP I, SPII, Xp) have been identified as cis regulators for transcription of the 3.5-, 2.4-, 2.1-a
nd 0.8-kb mRNAs, respectively. Two regions(nt1074 to 1234, nt1627 to 1774) in the HBV genome have been shown to act as transcriptional enhancers, Enhancer I (EN I )and Enhancer II(EN II), which show highly conservative trait, A lot of hepatocyte-specific factors bind to those two regions. For instance, C/EBP, HNF-4 and HB1F bind to EN I region, and the transcriptional factors, like C/EBP, HNF-4, HLF, HNF3, FTF and E4BP4, bind to EN II region. Only in the hepatocyte cells can these transcriptional factors be detected, but not in nonhepatic cells. This suggests that these transcriptional factors are necessary for the hepatocyte-specific traits of the EN I and EN II, and the hepatotropism of HBV replication might be related with these transcriptional factors.
Therefore we use the transcriptional regulatory sequence which might be attributable to the hepatotropism of HBV and use the therapic genes which only inhibit or kill tumor cells to construct the hepatic gene therapy vectors. When transferred into body, they will kill the hepatic tumor cells without damaging the normal cells. So we carry out our studies as follows: (1) Cloning of different HBV promoters and assessing of transcriptional activity of HBV promoter in hepatocyteor nonhepatic cells. (2) Construction of apoptin, antisense hTRT, IL-24 and
oncostatin M(osm) eukaryotic expression vectors driven by the HBV promoter and study of its expression specificity and anticancer effect in vitro and in vivo.
The preliminary research results as follows:
I. Construction of hepatocyte-specific vectors
1. Five Luciferase reporter plasmids driven by HBV EN II with basic core promoter(BCP), EN I and EN II with BCP, HBV EN II and BCP with mCMV promoter were successfully constructed.
2. A high hepatocyte-specific expression vector was achieved
The recombinant plasmids were transfercted into five hepatic cells(HepG2, 2.2.15, BEL-7402, SMMC7721, LO2) and five nonhepatic cells(HeLa, H460, MCF7, 803, A375)using Lipofectamine?reagent, The Luciferase expression which indirectly represented the transcriptional activeity of HBV promoter lying upstream of Luc gene was detected with Dual-Luciferase Reporter Assay System. the SV40 enhancer/promoter (pGL3-Control) as a positive control, and the negative control without promoter (pGL3-Basic). The Luciferase activity of pGL3-Control plasmid in each cell line was considered as 100%. The HBV promoter construct showed significant transcriptional activity in all hepatic cell lines., the transcriptional activity of EN IIR (mutant type) promoter was 0.987-5.79 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of EN I -IlW(ayw type) promoter was 1.25-15.31 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of EN I -IIR promoter was 3.76-21.83 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of ENIImCMV promoter was 9.49-91.71 fold of positive control under SV40 promoter in hepatic cell lines
引文
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2 Lehrman S. Virus treatment questioned after gene therapy death. Nature, 1999,401(6753):517-524
3 Su H, Lu R, Ding R, et al. Adeno-associated viral-mediated gene transfer to hepatoma: thymidine kinase/interleikin 2 is more effective in tumor killing in non-ganciclover(GCV)-treated than in GCV-treated animals. Mol Ther, 2000, 6:509-551
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5 Ido A, Uto H, Moriuchi A,et al. gene therapy targeting for hepatoceilular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human AFP.Cancer Research, 2001,61:3016-3023
6 Ohguchi A, Nakatsukasa H, Higashi T. et al. Expression of α-Fetoprotein and albumin genes in human hepatocellular carcionmas: limitations in the application of the genes for targeting human hepatocellular carcinoma in gene therapy. Hepatology, 1998, 27(2): 599-605
7 顾建人．新型基因转移系统在人肝癌基因治疗中．中国肿瘤生物治疗杂志，1999，6(3)：166-169
8 Rashid A, Wang JS, Qian GS. et al. Genetic alterations in hepatocellular carcinomas: association between loss of chromosome 4q and P53 gene mutations. British Journal of Cancer ,1999, 80(1/2):59-63
9 Anderson SC, Johnson DE, Harris MP, et al. p53 gene therapy in a rat model of hepatocellular carcinoma: intra-arterial delivery of a recombinant adenovirus. Clin Cancer Res, 1998,4:1649-1659
10 Huang X, Zhang W, Wakimoto H, et al. Adenovirus-mediated tissue-specific cytosine deminase gene therapy for human hepatocellular carcinoma with different AFP expression levels. J Exp Ther Oncol,2002,2(2): 100～106
11 Cao G, Kuriyama S, Du p, et al. complete regression of established murine hepatocellular carcinoma by in vivo tumor necrosis factor a gene transfer. Gastroenterology, 1997,112:501-510
12 Lin SB, Hsieh S H, Hsu HL, et al. Antisense oligdeoxynucleoxy nucleolides ofIGF-Ⅱ selectively inhibit growth of human heaptoma cells overproducing IGF-Ⅱ. J Biochem,1997,122(4):717-722
13 Kang MA, Kim KY, Seol JY, et al. The growth inhibition of hepatoma by gene transfer of antisense
vascular endothelial growth factor. J Gene Med, 2000,2:289-296
14 Barzon L, Bonaguro R, Castagliuolo I, et ai. Gene therapy of thyroid cancer via retrovirally-driven combined expression of human interleukin-2 and herpes simplex virus thymidine kinase. Eur J Endocrinol, 2003, 148(1):73-80
15 Sandig V, Loser P, Lieber A, et al. HBV-derived promoters direct liver-specific expression of an adenovirally transduced LDL receptor gene. Gene therapy. 1996, 3:1002-1009
16 Ishida H, Ueda K, Ohkawa K, et al. Identification of mμltiple transcription factors, HLF, FTF, and E4BP4,controlling hepatitis B virus enhancer Ⅱ.J Virol, 2000,74(3):1241-1251.
17 Fu L, Wu X, Kong YY, et al. Regulation of Hepatitis B Virus Gene Expression by Its Two Enhancers. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 1996,28(6):590-599.
18 Cai YN, Zhou Q, Kong YY, et al. LRH-1/hB1F and HNF1 synergistically up-regulate hepatitis B virus gene transcription and DNA replication. Cell Res. 2003,13(6):451-458.
19 Li M, Xie Y, Wu X, Kong Y, et al. HNF3 binds and activates the second enhancer, ENⅡ, of hepatitis B virus. Virology, 1995,214(2):371-378.
20 Nakamura TM, Morin GB, Chapman KB,et al. Telomerase catalytic subunit homologs from fission yeast and human. Science,1997,277:955-960
21 Avilion AA,Piatyszek MA,Gupta J,et al. Human telomerase RNA and telomerase activity in Immortal cell lines and tumor tissue. Cancer Res,1996,56(3): 645-653
22 Kondo S,Tanaka Y, Kondo Y, et al.Retroviral transfer of CPP32 geneinto malignant gliomas in vitro and vivo.Cancer Res,1998,58:962-973
23 Chen BG, Ray R, Dubik D et al. The E1B/bcl-2 binding protein NIP 3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med 1997,186:1975-1983
24 Zaring JM, Shoyab M, Marquardt H, et al. Oncostatin M:a growth regulator producted by differentiated histiocytic lymphoma cells.Proc Natl Acad Sci USA,1996,83:9739-9746
25 Bruce AG, Linsley PS, Rose TM, et al. Oncostatin M. Prog Growth Factor Res,1992,4:157-163
26 Jiang H, Lin JJ, Su ZZ, et al. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modμlated during human melanoma differentiation, growth and progression. Oncogene, 1995, 11(12):2477-2486
27 Mhashilkar AM, Schrock RD, Hindi M, et al. Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy. Mol Med, 2001, 7(4):271-282
28 aeki T, Mhashilkar A, Swanson X, et al. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene, 2002, 21(29):4558-4566
29 Galibert F, Mandart E, Fitoussi F, Tiollais P, Chamay E Nucleotide sequence of the hepatitis B virus geneome(subtype ayw) cloned in E.coli. Nature, 1979,281 (5733):646-50
30 J．萨姆布鲁克，D．W拉塞尔．分子克隆实验指南，科学出版社，2002年第三版
31 司徒镇强，吴军正．细胞培养，世界图书出版公司，1996年第—版
32 Kobayashi M, Koike K. Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization. Gene, 1984, 30(1):227-232
33 栾秋云，马张妹，张菁，成伟忠，闻玉梅．我国慢性乙型肝炎及肝癌患者中乙型肝炎病毒核心基因内缺失突变株的研究．上海医学．1999，22(10)：595-597
34 Li J, Buckwold VE, Hon M, et al. Mechanism of suppression of hepatitis B virus precore RNA transcription by a frequent double mutation. J Virol, 1999, 73(2): 1239-1244
35 Fang ZL, Yang JY, Wang XY, et al. The discovery ofHBV core promoter mutations in tumor tissue of patients with hepatocellular carcinoma. Virologica Sinica 2000, 15 (3) :244-248
36 王征旭，何振华，吴祖泽．腺病毒介导多基因对肝癌细胞生长的影响．中华普外基础与临床杂志，2000，7(6)：357-359
37 Sarkar D, Su ZZ, Lebedeva Ⅳ, et al. mda-7/(IL-24) Mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc Natl Acad Sci U S A, 2002, 99(15):10054-10059
38 Reymann D, Rousselle A V. gp130 cytokine family and bone cells. Cytokine, 2000, 12(10): 1455-1468
39 吕星，邢瑞云，路萍．OSM对黑色素瘤的抑制作用与基因-放射治疗效果．中国肿瘤生物治疗杂志，2001，8(1)：27-30
40 Mμller-Newen G The cytokine receptor gp130: faithfully promiscuous. Sci STKE, 2003 (201):1-3
41 Oro C, Jans DA. The tumour specific pro-apoptotic factor apoptin (Vp3) from chicken anaemia virus. Curr Drug Targets. 2004 5(2):179-90.
42 Pietersen AM, Vander E, Rademaker H J,et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene. Gene Ther, 1999, 6:882
43 谭芳，张玉静，徐彦波，刘万臣，张艳宇，柳凤琴．凋亡素(apoptin)诱导人白血病细胞的凋亡．中国兽医学报，2001，21(6)：571-572
44 孙国敬，童新，孙志贤．凋亡素选择性诱导肿瘤细胞凋亡及其分子机制．军事医学科学院院刊，1999，23(4)：299-301
45 Mμller-Newen G Ahmed A, Tollefsbol T. Telomeres, telomerase, and telomerase inhibition: clinical implications for cancer. J Am Geriatr Soc. 2003 Jan;51(1):116-22.
46 王智勇，鞠晓明，徐文怀，王振军，朱静．反义核酸hTRT对大肠癌细胞的抑制作用．北京大学学报(医学版)，2002，34(6)：692-695
47 杜辉，辛晓 燕，王健．hTETR基因反义核酸对8910卵巢癌细胞端粒酶活性的影响．第四军医大学学报，2000，21(3)：366—368
48 田凤军，王智勇，马俊义，赵云霞，卢炜．反义端粒酶逆转录酶对肺癌细胞的抑制作用．中华结核和呼吸杂志，2002，25(10)：635
2 Lehrman S. Virus treatment questioned after gene therapy death. Nature, 1999,401(6753):517-524
3 Su H, Lu R, Ding R, et al. Adeno-associated viral-mediated gene transfer to hepatoma: thymidine kinase/interleikin 2 is more effective in tumor killing in non-ganciclover(GCV)-treated than in GCV-treated animals. Mol Ther, 2000, 6:509-551
4 Teixeira LA, Fricke CH, Bonorino CB, et al. An efficient gene transfer system for the matopoietic cell line using transient and stable vectors. J Biotechnol, 2001, 88(2):159-165
5 Ido A, Uto H, Moriuchi A,et al. gene therapy targeting for hepatoceilular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human AFP.Cancer Research, 2001,61:3016-3023
6 Ohguchi A, Nakatsukasa H, Higashi T. et al. Expression of α-Fetoprotein and albumin genes in human hepatocellular carcionmas: limitations in the application of the genes for targeting human hepatocellular carcinoma in gene therapy. Hepatology, 1998, 27(2): 599-605
7 顾建人．新型基因转移系统在人肝癌基因治疗中．中国肿瘤生物治疗杂志，1999，6(3)：166-169
8 Rashid A, Wang JS, Qian GS. et al. Genetic alterations in hepatocellular carcinomas: association between loss of chromosome 4q and P53 gene mutations. British Journal of Cancer ,1999, 80(1/2):59-63
9 Anderson SC, Johnson DE, Harris MP, et al. p53 gene therapy in a rat model of hepatocellular carcinoma: intra-arterial delivery of a recombinant adenovirus. Clin Cancer Res, 1998,4:1649-1659
10 Huang X, Zhang W, Wakimoto H, et al. Adenovirus-mediated tissue-specific cytosine deminase gene therapy for human hepatocellular carcinoma with different AFP expression levels. J Exp Ther Oncol,2002,2(2): 100～106
11 Cao G, Kuriyama S, Du p, et al. complete regression of established murine hepatocellular carcinoma by in vivo tumor necrosis factor a gene transfer. Gastroenterology, 1997,112:501-510
12 Lin SB, Hsieh S H, Hsu HL, et al. Antisense oligdeoxynucleoxy nucleolides ofIGF-Ⅱ selectively inhibit growth of human heaptoma cells overproducing IGF-Ⅱ. J Biochem,1997,122(4):717-722
13 Kang MA, Kim KY, Seol JY, et al. The growth inhibition of hepatoma by gene transfer of antisense
vascular endothelial growth factor. J Gene Med, 2000,2:289-296
14 Barzon L, Bonaguro R, Castagliuolo I, et ai. Gene therapy of thyroid cancer via retrovirally-driven combined expression of human interleukin-2 and herpes simplex virus thymidine kinase. Eur J Endocrinol, 2003, 148(1):73-80
15 Sandig V, Loser P, Lieber A, et al. HBV-derived promoters direct liver-specific expression of an adenovirally transduced LDL receptor gene. Gene therapy. 1996, 3:1002-1009
16 Ishida H, Ueda K, Ohkawa K, et al. Identification of mμltiple transcription factors, HLF, FTF, and E4BP4,controlling hepatitis B virus enhancer Ⅱ.J Virol, 2000,74(3):1241-1251.
17 Fu L, Wu X, Kong YY, et al. Regulation of Hepatitis B Virus Gene Expression by Its Two Enhancers. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 1996,28(6):590-599.
18 Cai YN, Zhou Q, Kong YY, et al. LRH-1/hB1F and HNF1 synergistically up-regulate hepatitis B virus gene transcription and DNA replication. Cell Res. 2003,13(6):451-458.
19 Li M, Xie Y, Wu X, Kong Y, et al. HNF3 binds and activates the second enhancer, ENⅡ, of hepatitis B virus. Virology, 1995,214(2):371-378.
20 Nakamura TM, Morin GB, Chapman KB,et al. Telomerase catalytic subunit homologs from fission yeast and human. Science,1997,277:955-960
21 Avilion AA,Piatyszek MA,Gupta J,et al. Human telomerase RNA and telomerase activity in Immortal cell lines and tumor tissue. Cancer Res,1996,56(3): 645-653
22 Kondo S,Tanaka Y, Kondo Y, et al.Retroviral transfer of CPP32 geneinto malignant gliomas in vitro and vivo.Cancer Res,1998,58:962-973
23 Chen BG, Ray R, Dubik D et al. The E1B/bcl-2 binding protein NIP 3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med 1997,186:1975-1983
24 Zaring JM, Shoyab M, Marquardt H, et al. Oncostatin M:a growth regulator producted by differentiated histiocytic lymphoma cells.Proc Natl Acad Sci USA,1996,83:9739-9746
25 Bruce AG, Linsley PS, Rose TM, et al. Oncostatin M. Prog Growth Factor Res,1992,4:157-163
26 Jiang H, Lin JJ, Su ZZ, et al. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modμlated during human melanoma differentiation, growth and progression. Oncogene, 1995, 11(12):2477-2486
27 Mhashilkar AM, Schrock RD, Hindi M, et al. Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy. Mol Med, 2001, 7(4):271-282
28 aeki T, Mhashilkar A, Swanson X, et al. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene, 2002, 21(29):4558-4566
29 Galibert F, Mandart E, Fitoussi F, Tiollais P, Chamay E Nucleotide sequence of the hepatitis B virus geneome(subtype ayw) cloned in E.coli. Nature, 1979,281 (5733):646-50
30 J．萨姆布鲁克，D．W拉塞尔．分子克隆实验指南，科学出版社，2002年第三版
31 司徒镇强，吴军正．细胞培养，世界图书出版公司，1996年第—版
32 Kobayashi M, Koike K. Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization. Gene, 1984, 30(1):227-232
33 栾秋云，马张妹，张菁，成伟忠，闻玉梅．我国慢性乙型肝炎及肝癌患者中乙型肝炎病毒核心基因内缺失突变株的研究．上海医学．1999，22(10)：595-597
34 Li J, Buckwold VE, Hon M, et al. Mechanism of suppression of hepatitis B virus precore RNA transcription by a frequent double mutation. J Virol, 1999, 73(2): 1239-1244
35 Fang ZL, Yang JY, Wang XY, et al. The discovery ofHBV core promoter mutations in tumor tissue of patients with hepatocellular carcinoma. Virologica Sinica 2000, 15 (3) :244-248
36 王征旭，何振华，吴祖泽．腺病毒介导多基因对肝癌细胞生长的影响．中华普外基础与临床杂志，2000，7(6)：357-359
37 Sarkar D, Su ZZ, Lebedeva Ⅳ, et al. mda-7/(IL-24) Mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc Natl Acad Sci U S A, 2002, 99(15):10054-10059
38 Reymann D, Rousselle A V. gp130 cytokine family and bone cells. Cytokine, 2000, 12(10): 1455-1468
39 吕星，邢瑞云，路萍．OSM对黑色素瘤的抑制作用与基因-放射治疗效果．中国肿瘤生物治疗杂志，2001，8(1)：27-30
40 Mμller-Newen G The cytokine receptor gp130: faithfully promiscuous. Sci STKE, 2003 (201):1-3
41 Oro C, Jans DA. The tumour specific pro-apoptotic factor apoptin (Vp3) from chicken anaemia virus. Curr Drug Targets. 2004 5(2):179-90.
42 Pietersen AM, Vander E, Rademaker H J,et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene. Gene Ther, 1999, 6:882
43 谭芳，张玉静，徐彦波，刘万臣，张艳宇，柳凤琴．凋亡素(apoptin)诱导人白血病细胞的凋亡．中国兽医学报，2001，21(6)：571-572
44 孙国敬，童新，孙志贤．凋亡素选择性诱导肿瘤细胞凋亡及其分子机制．军事医学科学院院刊，1999，23(4)：299-301
45 Mμller-Newen G Ahmed A, Tollefsbol T. Telomeres, telomerase, and telomerase inhibition: clinical implications for cancer. J Am Geriatr Soc. 2003 Jan;51(1):116-22.
46 王智勇，鞠晓明，徐文怀，王振军，朱静．反义核酸hTRT对大肠癌细胞的抑制作用．北京大学学报(医学版)，2002，34(6)：692-695
47 杜辉，辛晓 燕，王健．hTETR基因反义核酸对8910卵巢癌细胞端粒酶活性的影响．第四军医大学学报，2000，21(3)：366—368
48 田凤军，王智勇，马俊义，赵云霞，卢炜．反义端粒酶逆转录酶对肺癌细胞的抑制作用．中华结核和呼吸杂志，2002，25(10)：635