【作者】 孙强玲；

【导师】 郑晓飞；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2004， 硕士

【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HB₁F，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 .76²1 .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%，pENHW-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更多还原

【Abstract】 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-and 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 andoncostatin 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 vectors1. 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 achievedThe 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更多还原