新型自发性人肺癌模型

Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths (around 13% of all deaths) in 2008. The most commonly diagnosed and most common cause of cancer death worldwide is the same cancer, lung cancer. In addition, lung cancer is of minimal change to be cured, with an overall 5-year survival rate of about 15%. Over decades, significant progress has been made in developing mouse models of lung cancer that could mimic the human disease. These models have been widely used for basic studies of tumor progression and gene function in vivo, as well as for translational studies of targeted therapies and drug resistance mechanisms. However, these models show limited similarities with the human disease. For example, the majority of murine lung tumor models develop relative benign adenomas and rarely more aggressive adenocarcinomas. The murine lung tumors exhibit limited vascularisation, and very few, if any, metastasize. A major reason for the discrepancies could be the differences between the two species. An emerging body of evidence indicates that subtle differences in the physiology of mouse and human can apparently cause fundamental differences in how the process of tumorigenesis occurs in the two species. And, of course, the differences can lead to different responsiveness to drug treatment. Therefore, we try to specifically knockout p53 and ectopically express K-RAS (G12D) at the same time in human embryonic stem cells (hESCs) derived alveolar type II cells (LT II). The in vitro differentiated LT II cells are transplanted back to the lung in SCID mice to investigate their potential tumorigenesis ability. Once lung cancer initiated from the transplanted LT II cells is defined in the mice, a novel spontaneous human lung cancer model is successfully developed. As hESCs is able to self-renew, unlimited amount of LT II cells derived from the genetically modified hESCs can be differentiated to make the novel model. Therefore, the novel human lung cancer model will be a powerful experimental system to investigate the molecular and cellular mechanisms under the tumorigenesis of human lung cancer, and to test the efficacy of new treatments against human lung cancer.

肺癌是发病病例和死亡病例最多的癌症，同时也是最难治愈的癌症之一，严重威胁着人类健康。虽然，现今有各种肺癌小鼠模型，极大地丰富了我们对于肺癌的发生和发展的认识。但这些模型也有明显的局限性。譬如，其恶性程度不高，血管生成较少，肿瘤转移能力差等。究其原因，很重要的一点是，小鼠细胞和人类细胞之间存在其固有的生理上的明显差异。这种差异同样会导致小鼠模型和病人的药效学不同。对此，我们拟对人类胚胎干细胞（hESCs）进行基因修饰，实现p53和K-RAS(G12D)分别在二型肺泡细胞（LT II）中特异性敲除和表达。体外分化这种hESCs成LT II细胞，将其移植到免疫缺陷型小鼠肺部，研究其致肺癌的能力，进而得到新型自发性的人肺癌模型。hESCs具有无限自我更新的能力，可无限量提供肺泡细胞来高重复性地建立这种模型。因此，一旦此模型构建成功，将成为我们研究人肺癌发生机制和验证新的治疗方法疗效的重要工具。

肺癌是发病率和死亡率都很高的癌症，严重威胁人类健康。当前使用的各种肺癌小鼠模型丰富了我们对肺癌发生发展的认识，但由于物种差异，小鼠模型不能全面重现人类肺癌的多种特征，包括恶性程度不高，血管生成少，肿瘤转移差等。在本课题中，我们根据TCGA （The Cancer Genome Atlas）数据库和文献筛查，挑选出可以促进肺癌发生发展的基因高突变位点和异常表达基因，包括KRAS（G12D），EGFR（L858R），P53缺失，PTEN缺失和c-Myc高表达。在人类胚胎干细胞（hESC）中，重建出相应的基因敲除和基因敲入的细胞株，其中P53和PTEN为诱导性敲除，c-Myc为诱导性高表达。利用3D类器官（organoid）技术，我们成功开发出一种体外体内结合的分化方法，可以高效率分化出I型和II型肺泡细胞（AT1和AT2细胞）。与已经发表的分化方法相比，我们的方法可以得到更多相对更成熟的AT1和AT2细胞。我们将各种突变细胞株进行肺部细胞分化，在体外和体内评估其成瘤性。为解决胚胎干细胞肺分化的难题，我们开发了多种基因组编辑方法来追踪和控制细胞分化的进程。具体为：基于CRISPR/Cas9和CRISPR/Cpf1技术，开发出hESCs中的大片段基因敲入方法，开发出基因组位点活体标记方法，开发出Brainbow四色荧光标记方法，开发出内源性基因激活方法。我们开发出的这些新方法不仅仅局限在细胞分化这一应用，其还具有更广泛的用途和价值。其中基因组位点活体标记技术可以应用于基因组3D结构研究，内源性基因激活技术可以一定程度上替代过表达系统，而可单独诱导或同时诱导的多基因敲除和多基因内源性基因激活技术是一门基础性的技术，有望广泛应用于细胞生物学各个领域。因此，通过本课题的研究，我们不仅有望建立人肺癌新模型，同时也开发出几种基础性的技术，对更广泛的生物学研究和医学研究提供有益的帮助。