转分化来源的肝干细胞载体介导靶向抗血管生成基因治疗肝癌的实验研究

BACKGROUND ＆AIMS The prognosis of patients with hepatocellular carcinoma (HCC) remains poor. The treatment still requires the development of novel therapeutic approaches. According to plasticity of peripheral hematopoietic stem cell and selective tropism of liver stem cells targeted HCC lesions, the research was designed. This study was undertaken to explore the possibility and mechanism of novel anti-angiogenic therapy of HCC by applying targeted gene therapy vector of liver stem cell transdifferentiated from peripheral hematopoietic stem cell. METHODS Firstly,peripheral hematopoietic stem cell was transdifferentiated into liver stem cell in certain conditions. Secondly,recombinant lentivirus was constructed for carrying human endostatin(hES) gene and genetic marker of enhanced green fluorescent protein (EGFP). Then, gene modification of liver stem cells from peripheral blood made hES protein expressed efficiently to acquire antiangiogenic gene therapy vector cells which were the carriers of ES and tracer cells with EGFP. Related experiments in vitro and in vivo were performed to observe whether the gene therapy vectors of liver stem cell from peripheral blood kept the ability of targeting HCC and potential mechanism of tropism, and to reveal the therapeutical effects on inhibition of HCC growth and metastasis. EXPECTED RESULTS (1) New liver stem cells were acquired from transdifferentiation of peripheral blood stem cells , which were named liver stem cells from peripheral blood (LSC-PB). This new cell line displayed a similar capacity of proliferation and differentiation traits were not affected. The lentivirus vector was constructed and recombinant lenntivirus was generated in HEK 293T packaging cell line,which could introducted hES and EGFP gene into LSC-PB cells. New gene-vector stem cells, named LSC-PB-hES, were established, from which cells could express hES and EGFP stably. (2)Secreted hES inhibited the proliferation and induced apoptosis of vascular endothelial cells HUVEC in vitro, which was expressed in the supernatant of LSC-PB-hES ,with the features of concentration dependent. (3) LSC-PB-hES cells still showed the trait of selective tropism for HCC lesions,localization and proliferation in tumor tissue,and expressed hES and EGFP stably in vivo. Some chemotactic factors could be related to the mechanisms of tropism of LSC-PB-hES. In HCC tumor burdened nude mouses,LSC-PB-hES cells expressed hES and mediated antiangiogenesis effects,specifically targeting suppression of tumor cell proliferation,inducing apoptosis, thus inhibiting tumor growth and metastasis. EXPECTED CONCLUSIONS This research provided the primary theoretical supports that peripheral hematopoietic stem cell could turn into liver stem cell as a vector of targeted gene therapy of HCC, mediating novel anti-angiogenic treatment, so as to solve the difficulties of limited source of liver stem cell and lacking ideal carriers in targeted gene therapy of HCC.

如何提高原发性肝癌的疗效依然是一个严峻的挑战，肝癌的治疗需要进一步理论创新和技术突破。利用造血干细胞可塑性和肝干细胞靶向肝癌病灶的趋化特性，将外周造血干细胞在一定条件下转分化为肝干细胞，构建慢病毒载体将目的基因人血管内皮抑素和标记基因增强型绿色荧光蛋白转导至该肝干细胞，使其高效表达分泌人血管内皮抑素蛋白，成为介导抗血管生成基因治疗肝癌的载体细胞和示踪细胞，进行体内外相关实验研究，阐明外周血来源肝干细胞治疗载体靶向肝癌的能力和机制，揭示其介导血管内皮抑素靶向抗肝癌血管生成、抑制肿瘤细胞增殖、诱导凋亡，从而抑制肿瘤生长和转移的治疗作用，为外周造血干细胞转分化为肝干细胞作为载体，介导独特的靶向抗血管生成基因治疗肝癌奠定基础，以期解决目前肝干细胞来源困难、肝癌靶向基因治疗缺乏理想基因转移载体的困境，初步建立以转分化来源肝干细胞为载体靶向基因治疗肝癌的治疗策略体系。

如何提高原发性肝癌疗效依然是一个严峻的挑战，肝癌治疗需要理论创新和技术突破。当前肿瘤基因靶向治疗仍缺乏理想治疗载体。干细胞研究是生命科学领域的热点，根据课题组前期实验肝干细胞是肝癌靶向基因治疗潜在的理想载体。成体肝干细胞获取困难，如以外周造血干细胞转分化为肝干细胞作为细胞载体靶向基因治疗肝癌能取得预期效果，将为肝癌基因治疗提供一种全新的治疗模式和理念。. 本项目利用造血干细胞可塑性和肝干细胞靶向肝癌病灶的趋化特性，将外周造血干细胞在一定条件下转分化为肝干细胞，构建慢病毒载体将目的基因血管内皮抑素和标记基因增强型绿色荧光蛋白转导至该肝干细胞，使其高效表达分泌人血管内皮抑素蛋白，成为介导抗血管生成基因治疗肝癌的载体细胞和示踪细胞，进行体内外相关实验研究，阐明外周血来源肝干细胞治疗载体靶向肝癌的能力和对肝癌生长和转移的抑制作用，并探讨其相关机制。 研究表明：体外可诱导外周血造血干细胞转分化为肝干细胞,利用分泌型人内皮抑素（Endostatin）基因过表达慢病毒转染，获得表达Endostatin的治疗性肝干细胞LSC-PB-ES。在体外实验中观察到细胞上清液中存在Endostatin表达，可抑制人血管内皮细胞ECV304增殖（P＜0.01），导致凋亡（P＜0.05），而对肝癌细胞株HCCLM3增殖无直接影响。动物实验中观察到，裸鼠尾静脉定期注射外周血干细胞来源的载体治疗细胞，携带绿色荧光EGFP的载体细胞对肝癌皮下移植瘤和转移瘤有靶向作用。实验组中裸鼠皮下移植瘤生长受到抑制不明显，但裸鼠肝、肺转移发生率和转移瘤的数量下降（P＜0.05），肿瘤及瘤周围表达endostatin增加，血管密度明显下降（P＜0.05），达到阻抑肝癌转移的有效作用。而脐血干细胞转分化来源的肝干细胞同样可以抑制裸鼠荷瘤鼠的肝、肺转移。. 项目的科学意义在于，探索了转分化不同来源肝干细胞治疗载体制备，明确了转分化来源的肝干细胞，基因改造后作为抗血管生成的治疗细胞，对实体肝癌原发灶有一定抑制作用，但主要通过抑制肿瘤血管生成，从而阻抑肝癌转移，可望同传统化疗结合达到靶向治疗的协同作用。转分化来源的肝干细胞作为载体，介导独特的靶向抗血管生成，为基因治疗肝癌奠定基础，可以初步解决目前肝干细胞来源困难、提供较理想基因转移载体，初步建立以转分化来源肝干细胞为载体靶向基因治疗肝癌的新策略。