腺相关病毒（AAV）基因靶向载体定点整合诱导肝癌的机制及相关研究

Many kinds of animal models have been widely used to study human hepatocellular carcinoma (HCC) pathogenesis and biological characteristics. Existing animal cancer models are limited in their ability to induce the process of carcinogenesis similar to human primary HCC, and to induce the high incidence and reproducibility. Previously report revealed that mice that received a randomly integrated adeno-associated virus (AAV) vector developed HCC that contained integrated vector proviruses mapped to the same 6 kb imprinted region on chromosome 12, suggesting that these particular integration events somehow led to HCC. In the human genome, the same imprinted genes are located in chromosome 14 q32 region, this region contains a large number of microRNAs and snoRNAs. These small RNAs could regulate a large number of target genes, and the human homologues of some of these microRNAs have been proposed to both stimulate and inhibit tumorigenesis in other types of malignancies. This provides a new research direction for us to study the mechanism driving hepatocarcinogenesis. Here we propose to follow up on our prior results in this area, we design a novel method for generating tumors through in vivo gene targeting Rian locus with AAV vectors, which introduces a specific chromosomal mutation and activate imprinted domain of chromosome 12 that is not normally expressed in adult hepatocytes. Pilot study reveal that normal newborn mice that received an intravenous injection of an AAV gene targeting vector nearly 100% developed HCCs. By using this animal model,we will study the role of this locus in HCC formation and development, how to activate/ intiate oncogenes and inactivate tumor suppressors, their gene expression patterns and their similarity to human HCC. The approach employed in this study is an especially attractive platform which may allow specific tumors to be studied in large animals that cannot otherwise be genetically manipulated, and provide outstanding models for studying the mechanism driving hepatocarcinogenesis and developing human anti-cancer treatments and drug screening.

动物模型是研究人类肝癌发病机制及生物学特性的主要工具。虽然已开发了许多动物模型来研究肝癌，但缺乏诱癌过程相似于人类原发性肝癌的发生过程以及诱癌率高和重复性好的动物模型。先前的研究结果显示，给予小鼠随机整合AAV载体可诱发肝癌，并从肿瘤中分离出整合的AAV前病毒在小鼠第12号染色体上qF1印迹区域。我们推测这个印迹区域可能与肝癌发生有关。在人类基因组中，相同的印迹基因组位于第14号染色体q32区域，这个区域包含了大量的miRNA和snoRNA，这为研究肝癌的发生机制提供了新的研究方向。我们利用体内基因打靶的方法去诱导特定染色体突变导致细胞转化并发展成特定的肿瘤。我们设计了一个AAV靶向载体，插入在Rian基因位点。初步的结果显示，正常新生小鼠接受AAV靶向载体，几乎全部诱发肝癌。我们将利用这个新的肝癌动物模型去研究肝癌的发生机制以及生物和药物干预、药物筛选，以及它们与人类肝癌关系。

动物模型是研究人类肝癌发病机制及生物学特性的主要工具，但目前仍缺乏诱癌过程相似于人类原发性肝癌的发生过程以及诱癌率高和重复性好的动物模型。我们利用AAV打靶技术将CAG增强子/启动子原件特异性的插入到了小鼠第12染色体qF1区域的Rian基因的第2内含子区域内，接受AAV靶向载体的小鼠几乎全部发生肿瘤，而且雄性小鼠比雌性小鼠发生肿瘤时间早及肿瘤的进展更加严重。对肿瘤组织进行组织病理分析可见典型肝细胞肝癌表现，BrdU的摄取明显增多。伴随着小鼠肝脏肿瘤的发展，小鼠外周血胆红素和肝酶的水平增加，白蛋白、总蛋白的水平降低。我们研究表明每个肿瘤细胞中至少含有一个AAV载体拷贝数的整合位点，AFP免疫组化分析显示AAV靶向载体诱发了单个细胞癌变进而发展为大的肿瘤结节，此外我们还发现EpCAM、PI3K、mTOR、P38 MAPK、CD31在肿瘤中高表达，初步探索了与肿瘤发生有关的信号通路。用AAV诱导小鼠肝癌的模型，诱癌率高, 实验重复性好，为人为诱发肝癌开辟了新的途径，为肝癌新药研发、筛选、评价提供了优良的模型，同时我们还发现了Rian基因在肝癌病理发生中的作用。实验还验证了AAV载体的基因毒性，为临床使用AAV载体基因治疗的潜在风险提供非常有价值的资料。

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