抗肿瘤干细胞及肿瘤血管生成的肝脏靶向性纳米基因治疗

Hepatocellular carcinoma (HCC)is a severe life-threatening disease for human. Anti-angiogenisis gene therapy has evolved as a promising treatment for HCC. However, a liver cancer cell targeting gene delivery vector is lacking. Previously, we developed a polymeric nanoparticle consisted of a co-polymer(H1) and plasmid DNA. H1 nanoparticle can safely and effectively tranduces genes into tumor cells both in vivo and in vitro. Here, we showed that when injected by tail vein or peritoneal, the H1 nanoparticles efficiently express target genes in the liver. When a CpG-free plasmid was used to express gene in the nanoparticles, the expression of the reported gene continually exceeded 45 days. Furthermore, we used H1 to deliver CpG-free plasmid encoding anti-angiogenisis genes, Endostatin and the kringle 1 domain of human HGF α-chain (HGFK1), to treat liver cancer in an orthotropic HCC mice model. We demonstrated that the single H1-mediated Endostatin (H1/pEndo, DNA 100ug), the single H1-mediated HGFK1(H1/pHGFK1, DNA 100ug) and combination of H1/pEndo(DNA 50ug) and H1/HGFK1 (DNA 50ug), all suppressed the tumor growth and prolonged the survival of mice significantly in the orthotropic mouse HCC model. Importantly, the combination treatment of H1/pEndo and H1/HGFK1 showed the best anti-tumor effects. We found that the combination treatment not only significantly reduces the amount of the micro vascular density (MVD), but also decreases the number of liver cancer stem-like cells in the tumor tissue. Based on these results, we propose to further study the mechanism underlying the anti-cancer stem cell effect. In addition, we will elucidate the functions of cancer stem cells on angiogensis and vasculogenic mimicry of the tumor. The liver-targeting H1 nanoparticle could be potentially developed to carry combined Endostatin and HGFK1 genes for the effective treatment of HCC.

原发性肝细胞癌是一种严重威胁人类健康的疾病。抗血管生成基因治疗策略被认为有希望治疗肝癌。但是存在两个问题：一是缺乏安全、有效的和肿瘤靶向性的基因药物传递载体；二是肿瘤血管的发生有多种机制，包括内皮细胞依赖的血管生成和血管生成拟态，因此，单独的一种抗血管生成基因并没有产生显著的临床利益。最近，我们发展了一种低毒、高效并能靶向性传递治疗基因到肝脏组织的非病毒基因药物传递系统（PEI-CyD-FA,命名为H1），而且单次注射，转导基因可持续表达超过40天。我们使用H1转导Endostatin和HGFK1基因，在小鼠原位肝癌模型上产生了明显的抗肿瘤作用。进一步我们发现，其治疗功效直接相关于抑制肝脏肿瘤血管内皮细胞依耐性血管生成和肿瘤干细胞。我们申请此项目资助，拟解决其抑制肿瘤干细胞的分子机理，探讨肿瘤干细胞在血管生成拟态及内皮细胞依耐性血管生成中的功能和作用，并进一步探讨上述药物的临床应用相关性。

肝癌干细胞是肝癌转移、复发和化放疗抵抗的“元凶”，发展靶向性抑制肝癌干细胞的药物，是亟待解决的问题。项目负责人的前期研究证明，腺相关病毒转导HGFK1基因能抑制肝癌的增殖和抑制肿瘤血管生成。然而，由于病毒载体的生物安全性和免疫活性限制，使其很难进入临床。在此项目资助下，课题组发展了一种高效、低毒、并具有肝脏靶向的纳米基因载体技术（H1/pCpG-free）；使用该技术，课题组证明：.1、腹腔注射H1/pHGFK1，H1/pHGFK1+H1/pEndo纳米基因粒子，有效抑制小鼠原位肝癌模型的肿瘤生长，并显著延长嗬瘤鼠存活时间；.2、H1/HGFK1，H1/Endo及两者联合应用抑制肿瘤血管生长并减少CD90+肝癌肿瘤干细胞数目；.3、Endostatin和HGFK1均能抑制血管内皮细胞增殖并抑制血管内皮细胞的成管能力，两种联合使用，协同性增加其功能；.4、2.6 HGFK1能抑制肝癌细胞干性，而Endostatin轻度减少其干性，两者联合能协同增强对并肝癌细胞干性的抑制功能.5、rHGFK1和rHGFK1+Endostatin均抑制了肝癌肿瘤干细胞的成球能力，并促使悬浮生长的肝癌肿瘤干细胞贴壁；然而，单独的Endostatin却不影响肿瘤干细胞的成球能力。.6、HGFK1绑定Met受体，抑制Met/β-cantenin信号轴调控肝癌干细胞干性。.这些研究说明：.1、成功发展了一种高效、低毒和肝脏靶向性纳米基因药物（H1/pCPG-free）传递技术；.2、证明H1/pEndostatin，H1/pHGFK1和H1/pEndostatin+pHGFK1纳米粒子，通过抑制肝癌血管生成和肝癌干细胞发挥抗肝癌效果；其中， HGFK1和Endostatin+HGFK1能有效抑制肝癌干细胞的自我更新，并促进其分化；.3、HGFK1可能是通过抑制Met/β-cantenin信号轴而调控肝癌干细胞活性。. 4、H1/pHGFK1和H1/pEndostatin+pHGFK1是一种既能抑制肿瘤血管生成，也能抑制肿瘤干细胞的纳米基因药物，具有很大的临床应用价值。