【作者】 刘新建；

【导师】 张大兵； 黄倩；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 目的:恶性肿瘤严重危害人类健康,基因治疗给肿瘤治疗带来了新的希望。肿瘤基因治疗涉及如何将治疗基因有效地送达靶组织和靶细胞,调控治疗基因选择性、高效地在肿瘤细胞内表达,以最大限度杀伤肿瘤细胞又不损害正常细胞作为发展方向和终极目标。由于肿瘤选择性复制型腺病毒载体(又称溶瘤腺病毒载体)可以选择性地在肿瘤细胞内复制并裂解肿瘤细胞,其携带的治疗基因也伴随病毒的复制明显增加,可以明显提升肿瘤基因治疗的效果。本研究拟构建一种新型的对肿瘤细胞感染效率更高、能够选择性在wnt信号异常肿瘤细胞内复制、携带治疗基因的嵌合型溶瘤腺病毒,并对这种新型重组腺病毒的抗肿瘤活性、动物体内分布特征等进行观察和分析。方法:5型、11型腺病毒分别以CAR、CD46为初级受体,在不同组织中CAR、CD46受体表达水平各有差异。为此,采用免疫组化和Real-time PCR方法检测了人正常组织及肿瘤组织表面CAR和CD46分子的表达水平。采用luciferase活性检测分析了5型腺病毒载体和外壳为Ad11b纤维蛋白、核心为Ad5的嵌合型腺病毒载体(Ad5-Luc和Ad5F11b-Luc)对肿瘤细胞的感染效率和外源基因表达水平。小动物活体成像比较荷瘤鼠瘤内注射或正常裸鼠尾静脉注射Ad5和Ad5F11b在体内的表达效率、生物分布以及血液清除率特性。wnt信号通路的关键调控点--β-catenin基因(CTNNB1)通过与下游基因启动子区的TCF/LEF结合位点结合上调相关基因的表达。检测分析β-catenin在肿瘤组织和细胞中是否存在突变与表达异常。利用含TCF/LEF结合位点重复序列(本文简称wnt启动子)的M50 Super 8X TOPFlash质粒转染细胞,分析β-catenin基因突变与wnt信号过度活化的关系;构建以wnt肿瘤特异性启动子调控腺病毒E1A基因表达,携带mda-7/IL24 Ad5F11b嵌合型溶瘤腺病毒( Ad5F11b.wnt-E1A-hIL24 )及不含mda-7/IL24的Ad5F11b.wnt-E1A对照病毒。新型重组腺病毒的抗肿瘤效果采用一系列体外实验予以证实,包括MTT、结晶紫染色、ELISA、Annexin V染色等;Western blot检测分析细胞凋亡途径,探讨其作用机制;通过荷瘤鼠治疗实验评价了新型溶瘤腺病毒在体内的抗肿瘤效果。结果:免疫组化检测到CAR在食道上皮细胞、胃上皮细胞、前列腺细胞中高表达,在肠上皮细胞、心肌细胞中中度表达,在肝脏、肺组织中低表达;Real-time PCR定量检测到CD46分子在13例结直肠癌和17例胃癌肿瘤中都有不同程度的升高,而CAR在大部分肿瘤(12例结直肠癌;10例胃癌)中表达下调。Luciferase活性检测显示感染Ad5F11b-Luc的细胞Luciferase的活性普遍比感染Ad5-Luc的细胞高。小动物活体成像显示,瘤内注射Ad5F11b-Luc后Luciferase在肿瘤内的表达强度明显比Ad5-Luc强,持续时间明显比Ad5-Luc长;尾静脉注射Ad5F11b-Luc及Ad5-Luc后观察二者在小鼠体内的分布特征与清除情况,Ad5F11b-Luc较Ad5-Luc在小鼠肝脏内存留量少、存留时间短,二者也存在明显差异。这表明Ad5F11b对肿瘤细胞有较高的感染效率,而对正常小鼠的肝的感染效率较低,提示Ad5F11b毒副作用可能比Ad5低。PCR产物直接测序证实38.5%(5/13)的结直肠癌、47.1%(8/17)的胃癌肿瘤标本、四株受检的肿瘤细胞株(HepG2,BEL7402,HCT116/4016,HCT116/379.2)有β-catenin基因突变;wnt报告基因活性检测分析表明有β-catenin基因突变的四株肿瘤细胞其中三株(HepG2,HCT116/4016,HCT116/379.2)的β-catenin/TCF活性都明显增加。嵌合型溶瘤腺病毒体外实验表明, Ad5F11.wnt-E1A和Ad5F11b.wnt-E1A-hIL24能选择性靶向wnt信号异常的肿瘤细胞,并显著性抑制wnt信号异常肿瘤细胞增殖,具有较强的肿瘤细胞毒性,这种选择性细胞毒性与病毒剂量和感染时间呈正相关,表现出嵌合型溶瘤腺病毒的病毒治疗特性。Ad5F11b.wnt-E1A-hIL24的抗肿瘤作用比Ad5F11b.wnt-E1A强,与MDA-7/IL24蛋白表达及分泌有关,表现出MDA-7/IL24的基因治疗特性。Ad5F11b.wnt-E1A-hIL24的抗肿瘤作用表现为诱导肿瘤细胞凋亡,其作用机理可能与激活被感染的肿瘤细胞caspase级联反应有关。荷瘤鼠体内实验证实,瘤内注射Ad5F11b.wnt-E1A-hIL24能有效抑制肿瘤生长,延长荷瘤鼠的生存期。结论:人肿瘤中存在wnt信号通路异常激活,wnt信号异常途径可以作为抗肿瘤治疗的靶标之一;Ad11型的受体在人的肿瘤组织中高水平表达,Ad11能高效感染人的肿瘤细胞;以wnt信号异常为靶标构建的、携带有mda-7/IL24治疗基因的靶向性、嵌合型溶瘤腺病毒(Ad5F11b.wnt-E1A-hIL24)在体内外均能够有效抑制人肿瘤细胞生长,其作用与诱导细胞凋亡有关。这种新型溶瘤腺病毒为肿瘤基因治疗提供了新的思路。更多还原

【Abstract】 Objective: Cancer is a major public health problem around the globe, and gene therapy has shown promising results in preclinical cancer treatment in recent years. The challenge for cancer gene therapy will be to successfully delivery therapeutic gene to target tissue, and the therapeutic gene selectively, efficiency express in target tissue, which will help to achieve the goal of completely eradicating tumor cells with minimal toxicity to normal cells.Replication competent viruses, termed oncolytic viruses are essentially tumor-specific, self-replicating, and lysis-inducing cancer killer, and the replication of virus also increase the amount of therapeutic gene. For study more efficiency method of cancer gene therapy, we designed a novel chimeric oncolytic adenoviral vector armed with therapeutic gene for the treatment of aberrant wnt signaling cancer and show its higher infection efficacy, selectively replication in tumor cells, anti-tumor effect, and systemic biodistribution in vitro and in vivo.Methods: Ad5 requires the coxsackie-adenovirus receptor (CAR) on the cell membrane as a primary receptor for infection, while Ad11 requires CD46 as a primary receptor. The CAR, CD46 expression levels are different in normal tissues and tumors. Therefor, the expression levels of CAR, CD46 in different normal tissues and tumors were examined by immunohistochemistry or Real-time PCR. Infected cells with Ad5-Luc and Ad5F11b-Luc (a chimeric Ad5 with Ad11 fiber), the infection efficiency and transgene expression levels were assayed by Luciferase Assay System. Intratumoral injecting Ad5F11b-Luc or Ad5-Luc in mouse xenografts or intravenous injecting Ad5F11b-Luc or Ad5-Luc into normal nude mice, tumor local gene expression, systemic biodistribution and blood clearance properties of Ad5F11b and Ad5 vectors were analyzed by Live Cell Imaging System.To determine if aberrant wnt signaling in tumor cells can be used to selectively drive viral replication, we have analyzedβ-catenin mutation status of tumor tissues as well as cell lines. And we screened forβ-catenin/TCF-hyperactivated (aberrant wnt signaling) tumor cell by transfected M50 Super 8X TOPFlash plasmid which contain an luciferase expression cassette driven by 8 copies of TCF/LEF binding site (named wnt promoter). Based on these findings, a novel chimeric oncolytic adenoviral vector containing an E1A expression cassette driven by an artificial wnt promoter have been constructed to selectively replicate in aberrant wnt singaling tumor cells.For more increase the anti-tumor effect of this novel oncolytic adenovirus, an apoptosis inducing gene, interleucin-24 expression cassette driven by cytomegalovirus promoter was inserted into its E3 region. Therefore, a novel chimeric oncolytic adenoviral vector containing an E1A expression cassette driven by an artificial wnt promoter, pseudotyped fiber by replacing Ad5 with Ad11 Fiber and delivering mda-7/IL24 gene (Ad5F11.wnt-E1A-hIL24) or not (Ad5F11.wnt-E1A) was designed for treatment aberrant wnt signaling tumor. The therapeutic effect in vitro was determined by MTT, crystal violet staining, ELISA, Annexin V staining assay, and anti-tumor effect mechanism was evaluated by analysis the activation of caspase3, caspase7 and PARP using western blot assay. And the anti-tumor effect in vivo was evaluated in mouse xenograftsResults: We subjected 20 different normal human tissues to immunohistochemical analysis of CAR expression, the results showed that CAR expression high in esophagus and gastric epithelium, prostate; intermediate in intestinal epithelium, and myocytes of the heart; and low in liver, lung. Tumor tissues were subjected Real-time PCR analysis of CAR and CD46 expression, the results showed that CD46 expression levels were higher in almost all tested tumor than their adjacent normal tissues. In contrast, CAR expression levels were lower in almost all tested tumor tissues than their adjacent normal tissues (colorectal tumor:12/13; gastric tumor:10/17). The infection efficiency and transgene expression levels of Ad5F11b and Ad5 infected cells were determined in vitro using Luciferase Assay System, the results showed that the luciferase expression of Ad5F11b-Luc infected cells were significantly improved compared to non-pseudotyped Ad5-Luc control viruses. And the differential gene transduction of Ad5-Luc and Ad5F11b-Luc infected cells were further determined in vivo using Live Cell Imaging System, the results showed that the tumor local luciferase expression levels and time of intratumoral injection Ad5F11b-Luc were significantly increased compared to that of Ad5-Luc, and intravenous injection Ad5F11b-Luc exhibited a less amount and shoter-term in liver than that of Ad5. Due to the higher tumor local gene transduction and lower liver infection efficieny of Ad5F11b, it would be less side-effect than Ad5.Sequencing analysis ofβ-catenin mutations showed that five out of 13 (38.5%) colorectal and eight out of 17 (47.1%) gastric tumors showed point mutations in tumor tissues but not in their adjacent normal tissues. BEL7402,HCT116/4016, HCT116/379.2 also showed point mutation, but HepG2 showed deletion mutation. Cell lines carryingβ-catenin mutations (HepG2, HCT116/4016 and HCT116/379.2) exhibitedβ-catenin/TCF- hyperactivated activity compared to cell lines with wildtypeβ-catenin. Ad5F11.wnt-E1A, Ad5F11.wnt-E1A-hIL24 oncolytic adenovirus can selectively replicate in aberrant wnt signaling tumor cells, and displayed a powerful efficacy in killing aberrant wnt signaling tumor cells. This selectivity cytotoxicity is cosistant to the dose of virus and its infection time. And the anti-tumor effect of Ad5F11.wnt-E1A-hIL24 was significantly improved compared to Ad5F11.wnt-E1A due to the expression of MDA-7/IL24. The therapeutic effect was associated with increased apoptosis through caspase 3 activation. Intratumoral injection Ad5F11.wnt-E1A-hIL24 resulted in profoundly inhibition of tumor growth and longterm survival.Conclusions: Aberrant wnt signaling was identified in a variety of tumors and it can be introduced to targeting cancer gene therapy. The primary receptor of Ad11 high-level expresses in a variety of tumors and Ad11 can efficiently infect tumors. Our modified chimeric oncolytic adenoviruses expressing mda-7/IL24 (Ad5F11.wnt-E1A-hIL24) could exert potential anti-tumor activity and significant apoptosis in aberrant wnt signaling tumor and offer a novel approach to cancer gene-viral therapy.更多还原