【作者】 李军；

【导师】 窦科峰； 李海民；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 原发性肝癌是恶性程度最高的肿瘤之一,目前尚无根治方法。自杀基因疗法,又名基因介导的酶-前药疗法,能特异性提高药物在肿瘤中的浓度,并具备独特的旁观者效应,是肿瘤基因治疗的重要方法之一。本研究将木薯来源的linamarase(lis)引入肝癌基因治疗中,利用其水解前体药物linamarin(lin),并产生毒性代谢产物HCN的特性,从而实现对肝癌细胞的杀伤。通过建立lis稳转肝癌细胞系,发现其本身对细胞生长,细胞周期等特性并无影响。此外单独使用前体药物lin也未发现细胞毒性,从而提示了该策略的安全性。为提高目的基因的表达及针对肝癌的靶向性,我们以ViraPower?病毒载体系统为基础,分别构建CMV及AFP启动子转录的lis腺病毒载体。体外实验表明,含CMV启动子的腺病毒联合lin具备针对多种肿瘤细胞的强大抑制效应及旁观者效应。而含AFP启动子的病毒仅具备对AFP阳性肝癌细胞的特异性杀伤,从而体现了其针对肝癌的特异性和靶向性。动物实验提示lis能特异性表达于肝癌组织中,联合使用lin后能显著抑制肿瘤生长并延长动物存活时间。结果表明,lis/lin系统是一种潜在的肝癌基因治疗策略,值得进一步深入研究。目的:1.构建稳定转染lis基因的肝癌细胞系,实现lis酶在真核细胞中的活性表达,观察它对肿瘤细胞的体内、体外生物学特性的影响。初步了解lis基因及lin对肝癌细胞的作用;2.运用ViraPower?腺病毒载体系统,构建CMV启动子转录下的lis重组腺病毒载体,观察该病毒载体的感染效率及目的基因表达情况。应用重组病毒联合前体药物lin进行多种肿瘤细胞系的体内、体外治疗,探讨腺病毒载体介导的lis/lin系统的抑瘤效果及作用机理;3.克隆AFP启动子,检测其靶向转录活性。构建AFP启动子转录的lis重组腺病毒,通过体外实验及裸鼠荷瘤实验来检验该病毒前药体系针对AFP阳性肝癌细胞的特异性杀伤,实现lis/lin系统治疗原发性肝癌转录水平上的靶向调控。方法:1.PCR法从木薯cDNA中克隆并扩增lis cDNA,将该基因亚克隆至pcDNA3.1+质粒中,构建重组真核表达载体。脂质体转染及药物筛选相结合,构建稳定转染lis肝癌细胞系HepG2/lis。观察稳转细胞形态学变化,生长曲线,流氏细胞周期,裸鼠体内成瘤曲线等,并进行肿瘤细胞内lis酶活性分析。用不同浓度的lin培养HepG2细胞,观察lin本身对HepG2细胞生长的影响及细胞毒性;2.将lis cDNA亚克隆于ViraPower?腺病毒载体系统的入门克隆pENTRTM2B中,利用特异性LR反应将入门克隆中的lis导入腺病毒骨架载体pAd/CMV/V5-DEST中(含CMV启动子) ,获得重组病毒骨架载体pAd/CMV/lis。线性化骨架载体并转染293A细胞,包装重组腺病毒Ad-CMV-lis。扩增并纯化重组腺病毒,TCID50法测定它的滴度。利用含报告基因的腺病毒Ad-CMV-EGFP感染肿瘤细胞,摸索病毒的最佳感染复数。通过联合Ad-CMV-lis及前药lin进行多种肿瘤细胞系的体外及体内抑瘤实验,观查该系统的抑瘤活性及旁观者效应。用DNA片段化检测、Annexin-V/PI流式细胞分析、瘤组织病理学及TUNEL染色进一步探索lis/lin系统的抑瘤机理。3.RT-PCR法从HepG2(AFP+)细胞基因组中克隆并扩增AFP启动子,亚克隆入pEGFP-1质粒中,利用报告基因EGFP检测其特异性转录活性。用AFP启动子替换pcDNA3.1+质粒中的CMV启动子,然后将lis cDNA插入该质粒多克隆位点中,实现AFP启动子与lis的串联。通过构建病毒入门载体及LR反应,将AFP-lis序列定向克隆于pAd/PL-DEST腺病毒骨架载体中(不含启动子),获得骨架载体pAd/AFP/lis。将其转染293A细胞,包装重组腺病毒Ad-AFP-lis。重组病毒经扩增、纯化后,联合lin进行体内、体外抑瘤实验,验证该体系对AFP阳性肿瘤细胞的特异性及靶向性杀伤。结果:1.成功构建lis稳转肝癌细胞系HepG2/lis。发现它与空质粒转染组及空白对照细胞相比,在细胞形态,生长曲线,细胞周期分布,体内成瘤生长曲线等方面差异不显著(P>0.05)。HepG2/lis细胞给予lin后,能产生代谢产物HCN,并随lin浓度的增加而增多,提示lis酶在真核细胞中能以活性方式表达。此外单独使用lin对细胞生长并无抑制作用。2.成功构建重组腺病毒Ad-CMV-lis。联合应用lin后能在体内、体外有效抑制多种肿瘤细胞的生长,抑瘤作用与lin呈明显的时间、剂量依赖效应。肿瘤细胞治疗后经DNA片段化检测未发现典型的凋亡DNA ladder, Annexin-V/PI流式细胞分析提示经治疗后凋亡及坏死细胞均增高,但以坏死细胞增高为主。瘤组织病理学观察发现Ad-CMV-lis/lin治疗组中出现大量的坏死区,并发现较多的TUNEL阳性染色细胞。3.克隆并重组了276bp的AFP启动子。EGFP报告基因分析提示,该启动子具备AFP阳性肝癌细胞的特异性转录活性。成功构建AFP启动子转录下的重组腺病毒Ad-AFP-lis,实现了lis在AFP阳性肝癌细胞系中的高效特异性表达。体外及动物治疗实验均证明,该病毒联合lin治疗,能特异性抑制AFP阳性肝癌细胞系的生长,并延长动物存活时间,而对AFP阴性肝癌细胞系无杀伤效果。结论:1.植物来源的lis cDNA能稳定表达于真核细胞中,并能正确包装出类似大小,具备β-葡萄糖苷酶活性的lis蛋白。lis cDNA在细胞中的稳定整合对细胞形态、生长特性、细胞周期、成瘤性等生物学特点并无明显影响。而前体药物lin对细胞也具有低毒性,因此lis/lin系统是一种安全的自杀基因治疗策略。2.通过重组腺病毒载体,发现腺病毒具有靶细胞感染率及目的基因表达水平高,包装使用方便,病毒滴度高,不整合到宿主基因组中等优点,因此能作为lis/lin自杀基因治疗的高效表达载体。3.重组腺病毒Ad-CMV-lis在体内、体外均表现出对多种肿瘤细胞系的抑制作用,提示lis/lin自杀基因系统具备较广的治疗适用范围。由于不同肿瘤的生物学特点不同,因此该系统在其他肿瘤中的应用潜能有待于进一步研究。4. 276bp的AFP启动子能调控lis/lin系统在肝癌中的靶向性转录,实现针对AFP阳性肝癌细胞的特异性杀伤。然而由于原发性肝癌中有少部分呈AFP阴性表达,因此改造现有的AFP启动子,提高其针对AFP阴性的肝癌细胞的转录水平,是值得深入研究的问题。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignances worldwide, and there is no effective treatment for it until now. Suicide gene therapy, also known as gene-directed enzyme prodrug therapy, is an important strategy for tumor treatment by improving the specifical concentrations of drugs in tumors and a unique bystander effect. The research here presents a novel suicide gene therapy strategy against HCC based on the linamarase (lis), a gene from cassava, which can hydrolyze prodrug linamarin (lin) into HCN. Lis cDNA was stably expressed in HCC cells. Studies in vitro indicated that stable expression of lis had no direct effects on the proliferation and cell cycle of hepatoma cells. In addition, the administration of lin lonely is harmless too, indicating that the system is safe. In order to enhance target gene expression in HCC cells, we reconstructed adenoviruses involving lis with CMV or AFP promoter based on ViraPower? adenovirus system respectively. In vitro studies showed that Ad-CMV-lis combined with lin had strong antitumor effect and bystander effect on a variety of tumor cells. While Ad-AFP-lis with lin only showed inhibiting effect on AFP-positive hepatoma cells, which indicated the target specificity of the adenovirus to HCC. Animal experiments showed that lis was expressed highly in the tissue of tumors specifically , and significantly inhibited tumor growth and prolonged animal survival combined with lin. All findings suggest that lis/lin system may provide a practical and promising approach for the treatment of HCC and be worthy of further study.Objective:1. To construct a new hepatoma cell line which can stably express lis cDNA and evaluate the activity of lis enzyme expressing in eukaryotic cells. To observe the effect of lis on the biological characteristics of hepatoma cells in vitro and in vivo.2. At the base of ViraPower ? adenovirus system, to reconstruct an adenoviruse vector containing lis cDNA which is regulated by CMV promoter. To Evaluate the infective efficiency of the virus and the level of gene expression in cells infected by the adenovirus. The antitumor effect and the mechanism of the adenovirus mediated lis / lin system is explored in vitro and in vivo.3. To clone AFP promoter and detect its target transcription activity. Reconstruction of an adenovirus containing lis cDNA transcripted by AFP promoter. To test the inhibitory effect of the system against the AFP positive HCC cells by in vitro and tumor-bearing nude mice. Finally, achieving the targeted transcription regulation of lis / lin system in HCC.Methods:1. Lis cDNA was cloned and amplified from cassava cDNA by PCR method. the sequence was then subcloned into pcDNA3.1+ plasmid, an eukaryotic expression vector. HepG2/lis cell which was stablely transfected with lis plasmid was built by lipofectamine and drug screening. The characteristics of HepG2/lis was observed, including cell morphology, growth curve, cell cycle, tumorigenesis in nude mice and enzyme activity of lis. The toxicity of lin itself in cells was also observed by administrated HepG2 cells using different concentrations of lin;2. Lis cDNA was subcloned into the pENTRTM2B entry vector of ViraPower? system, and then delivered to pAd/CMV/V5-DEST destination vector (containing CMV promoter) by using specific LR reaction between th entry vector and the destination vector. The newly obtained pAd/CMV/lis destination vector was linearized and then transfected into 293A cells for packaging recombinant adenovirus. The recombinant adenovirus (named Ad-CMV-lis) was amplified and purified, and the titers of the virus was also determined by TCID50 method. Hepatoma cells was infected by Ad-CMV-EGFP which contained the EGFP reporter gene to explore the best MOI of virus infection. A variety of tumor cell lines was treated by Ad-CMV-lis and lin in vitro and in vivo to observe the antitumor activity and bystander effect of the system. The inhibitory mechanism of lis/lin system was further explored by a variety of detection, including DNA fragmentation detection, Annexin-V/PI flow cytometry analysis, tumor histopathology and TUNEL staining.3. AFP promoter was cloned by RT-PCR from the genome of HepG2 cells (AFP+), then subcloned into plasmid pEGFP-1. The specific transcriptive activity of AFP promoter was detected by using EGFP reporter gene analysis. CMV promoter of pcDNA3.1+ plasmid was replaced by AFP promoter, then lis cDNA was inserted into the multiple cloning sites of the plasmid, achieving the tandem of AFP promoter with lis. By construction of adenovirus entry plasmid and the LR reaction, the AFP-lis sequence was subcloned into the pAd/PL-DEST backbone vector (no promoter). 293A cells was transfected with pAd/AFP/lis plasmid to package recombinant adenovirus Ad-AFP-lis. The recombinant virus was amplificated, purified and titered. The targeting specificity and antitumor effect of the new adenovirus with lin to AFP-positive tumor cells were detected in vitro and in vivo.Results:1. Lis was successfully introduced to stable transgenic HepG2/lis cell line. There were no significant differences in morphology, growth curve, cell cycle distribution and in vivo tumor growth curve of HepG2/lis cells compared with those of the empty plasmid transfection group or blank control cells (P> 0.05). When lin was administered to HepG2/lis cells, HCN could be detected in the metabolites and the amount of it was increaseing with the increased concentration of lin, suggesting that the protein of lis expressed in eukaryotic cells can be enzyme activity. In addition, lin itself shown no inhibitory effect on cell growth.2. Recombinant adenovirus Ad-CMV-lis was successfully constructed. The inhibitory effect treated with Ad-CMV-lis and lin in a variety of tumor cells was significant in a time- and dose-dependent manner in vitro and in vivo. There was no typical DNA ladder detected by the DNA fragment assay in the tumor cells after treatment. Annexin-V/PI flow cytometry analysis showed that apoptosis and necrosis cells were all increased, but consisted mainly of necrotic cells. Histopathological observation of tumors found many necrotic area arising in the Ad-CMV-lis/lin treatment group, and a more positive TUNEL staining cells detected.3. The 276bp sequence of AFP promoter was cloned and then reorganized. EGFP reporter gene analysis showed that the promoter had specific transcription activity in AFP-positive HCC cells. The recombinant adenovirus Ad-AFP-lis transcripted under the AFP promoter was successfully constructed. By using this adenovirus, lis could be highly and specifically expressed in AFP-positive HCC cell line. Treatment experiments in vitro and in vivo have proved that the united lin/Ad-AFP-lis treatment can inhibit the growth of AFP-positive HCC cells, and prolong animal survival time, while the AFP-negative hepatoma cell line was free of inhibitory effect.Conclusion:1. Lis cDNA of plant origin can be stably expressed in eukaryotic cells and be correctly packaged to protein at a similar size, withβ-glucosidase activity. The stable integration of lis cDNA into cells had no significant impact in the cell morphology, growth, cell cycle, tumorigenesis and other biological characteristics. The prodrug lin also had low toxicity, so lis/lin system is a safe suicide gene therapy strategy.2.Through recombinant adenovirus vector, we found that adenovirus has many advantages, including efficient infection , a high level of gene expression, easy to packaging in high-titer, not integrated into the host genome. So it can be a more efficient expression vector for lis/lin suicide gene therapy.3. Recombinant adenovirus Ad-CMV-lis shown a wide range of inhibition of tumor cell lines in vitro and in vivo, suggesting that lis/lin suicide gene system has a wider scope of application. Due to the different biology characteristics in different tumor, the potential applications of this system to other tumors must be further studied.4. The AFP promoter of 276bp can regulate the targeting transcription of lis/lin system in HCC and achieve its target of AFP-positive hepatoma cell-specific destruction. However, due to negative expression of AFP in a small number of primary liver cancer, the transformation of existing AFP promoter, enhancement its transcription level to AFP-negative hepatoma cells, is worthy of in-depth study.更多还原