载阿帕替尼温敏纳米凝胶栓塞肝癌及抑制肿瘤血管新生的机理研究

Transcatheter arterial chemoembolization (TACE) is the first choice for the treatment of advanced liver cancer. However, there are still exist two defects to influence the long-term effect of TACE. On one hand, the angiogenesis of hepatocellular carcinoma (HCC) induced by hypoxia in tumor after TACE has been proved to play an important role in the malignant biological behavior of HCC, on the other hand, embolization of tumor peripheral blood vessels can also be a key factor affecting the curative effect of TACE. Our previous studies have showed that Apatinib mesylate, as a single-target inhibitor of vascular endotheliar growth factor receptor (VEGFR), can efficient inhibit tumor angiogenesis by targeting the VEGF/VEGFR pathway. Meanwhile the temperature sensitive nano-gel has an excellent peripheral vascular embolization effect.. Therefore, in this study we will combine the anti-angiogenic effect of apatinib with TACE treatment, as well as the characteristics of the temperature sensitive nano-gel to synthesize a new embolic agent –Apatinib-PIB. A new targeted drug delivery system would be developed through this research, which will be meaningful to improve the interventional treatment effect of liver cancer. The physical and chemical properties, biocompatibility, release characteristics in vitro and the anti-angiogenesis of this new embolic agent will be evaluated as well. The mechanism of inhibition of VEGF/VEGFR pathway was elucidated, and antiangiogenic properties in anoxic environment or not and the role in the treatment of liver cancer will be explored as well. We hope that our effort could provide a new therapeutic strategy for interventional therapy of liver cancer.

经肝动脉化疗栓塞（TACE）术是中晚期肝癌的首选治疗，但仍存在栓塞材料是否栓塞到肿瘤末梢血管及术后肿瘤缺氧，引起残存肿瘤血管新生导致肿瘤复发、转移这两方面的问题，也是影响TACE中远期疗效的关键因素。我们前期研发的温敏纳米凝胶具有优异的末梢血管栓塞性能；同时前期实验揭示阿帕替尼具有高效抑制VEGF/VEGFR通路从而达到抑制肿瘤血管新生，增加TACE疗效的作用。 .据此提出将阿帕替尼与温敏纳米凝胶结合起来，制备新型的具有药物缓释特性的负载阿帕替尼温敏纳米凝胶，将其应用于肝癌TACE治疗的实验研究，并探索分子靶向药物原位靶向给药新方式。本研究拟评价载阿帕替尼温敏纳米凝胶的理化性能、生物相容性、体外药物释放等特性；阐明其对VEGF/VEGFR通路抑制的可能机制；并着重探讨其在缺氧及常氧状态下的抗血管生成特性及其对肝癌的作用。为肝癌介入栓塞治疗提供新型载药栓塞剂及新的治疗思路。

经肝动脉栓塞术是中晚期肝癌重要的治疗方法，栓塞后肿瘤缺氧往往与肿瘤的复发和转移有关，是限制TAE治疗效果的关键因素。我们前期研发的温敏纳米凝胶具有优异的末梢血管栓塞性能；同时阿帕替尼具有高效抑制VEGF/VEGFR通路从而达到抑制肿瘤血管新生。本研究旨在观察载阿帕替尼温敏纳米凝胶经介入栓塞治疗后VX2肝癌的治疗效果及栓塞后抑制肿瘤血管生成的作用。.我们在体外和体内模拟了TAE前后肿瘤微环境的变化。在体内研究中，65只载VX2肝癌的家兔被随机分为5组进行治疗，分别为：载阿帕替尼温敏纳米凝胶(PA组)；温敏纳米凝胶(P组)；单纯碘油(I组);单纯阿帕替尼灌注(A组)；经肝动脉注射生理盐水(NS组)。其中阿帕替尼剂量为2mg/kg。然后观察肿瘤生长情况并记录载瘤动物的生存时间。检测各组动物微血管密度的表达情况。在体外实验中,我们在人脐静脉内皮细胞(HUVEC)与肝癌HepG2细胞系共培养的培养基中加入不同浓度梯度阿帕替尼，并分为常氧或缺氧组来模拟TAE前后肿瘤微环境。通过相关测定方法来探讨阿帕替尼的作用机制。.PA组肿瘤生长速度明显低于其他四组(P=0.000<0.01)，且生存时间显著延长（P=0.000<0.01），CCK-8法实验进一步证实阿帕替尼在缺氧环境下具有抑制肝癌生长的能力。免疫组化的结果显示载阿帕替尼温敏凝胶栓塞（PA）组的肿瘤标本CD31染色明显减少（P=0.00<0.01）。更深入的治疗机制研究我们发现阿帕替尼在缺氧环境下有着更强的抑制HUVEC小管形成以及及迁徙的能力，并随浓度的增加其抑制能力也越强。用阿帕替尼处理后，HUVEC中p-ERK，p-AKT，p-PI3K和p-P38的蛋白表达水平下调，并且在缺氧环境下抑制更强。.载阿帕替尼温敏纳米凝胶可作为一种有效的液体栓塞材料用于VX2肝癌的介入治疗。阿帕替尼通过温敏纳米凝胶靶向运输到肝癌组织，并实现药物在肿瘤局部的缓慢释放作用；通过下调PI3K-AKT、Raf-MEK-ERK和P38MAPK信号通路，阿帕替尼阻滞了介入栓塞后缺氧环境下引起的血管生成和迁徙，并具有良好的延长生存时间和抗肿瘤生长的效果。本研究有望为肝癌介入治疗提供新的靶点和治疗策略，并具有良好的应用前景。