以过表达CXCR4 的间充质干细胞为载体的骨肉瘤纳米磁流体靶向热疗

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis is the major cause for therapeutic failure and low survival rate of osteosarcoma. Magnetic fluid hyperthermia (MFH) is a new thermal therapy using nanotechnology and hyperthermia, and it is promising in treating malignant cancers. Previous studies performed by the applicant revealed that mesenchymal stem cells (MSCs) can target to osteosarcoma and its metastases. The chemokine receptors type 4(CXCR4) expressed by MSCs can bind to stromal cell-derived factor-1 (SDF-1) secreted by osteosarcoma cells, which will mediate migration of MSCs. This study will first investigate the influence of CXCR4 overexpression and superparamagnetic iron oxide nanoparticles (SPION) labeling on migration and proliferation of MSCs, and analyze targeting ability of labeled MSCs towards primary osteosarcoma and metastatic lesions. Then MSCs over-expressing CXCR4 will be used as vehicles carrying SPION to primary osteosarcoma and metastatic lesions, then external alternating magnetic field will be introduced to conduct targeted MFH for primary osteosarcoma and multiple metastatic lesions. The study will bring breakthrough to the therapy of osteosarcoma.

骨肉瘤是青少年最常见的原发恶性骨肿瘤，转移病灶的出现是骨肉瘤治疗失败以及低生存率的主要原因。磁流体热疗(MFH) 是将纳米技术和热疗技术相结合的一种新兴热疗方法，在肿瘤治疗领域具有较大的应用前景。申请人既往的研究表明间充质干细胞(MSCs)对骨肉瘤及其转移灶具有靶向性，其表达的趋化因子受体4（CXCR4）可与肿瘤细胞分泌的基质细胞衍生因子-1（SDF-1）结合，介导其迁移。本研究首先探讨CXCR4过表达和超顺磁性氧化铁纳米粒子（SPION）标记对MSC迁移和增殖的影响，分析标记的MSCs对骨肉瘤原发及转移病灶的趋向性。然后利用过表达CXCR4的MSC 对骨肉瘤的靶向性，将SPION 携带至骨肉瘤原发及转移病灶内，再施以外加交变磁场，实现对骨肉瘤原发灶及多发转移病灶的靶向热疗。本疗法有望为骨肉瘤的疗效带来突破。

骨肉瘤是最常见的原发恶性骨肿瘤。研究表明间充质干细胞(MSCs)对骨肉瘤及其转移灶具有靶向性，基质细胞衍生因子-1 (SDF-1) 及其受体趋化因子受体4 (CXCR4)在多种恶性肿瘤的发生和发展中起到重要作用。采用CCK-8或MTT、流式细胞术和Transwell检测分析CXCR4过表达对MSCs和超顺磁性氧化铁纳米颗粒 (SPION)标记对MSCs增殖和侵袭的影响，并检测shRNA介导的CXCR4敲减以及CXCR4抑制剂AMD3100联合雷公藤甲素处理对骨肉瘤细胞增殖、凋亡及侵袭的影响。通过Western blot检测PI3K/Akt/NF-κB通路相关蛋白及凋亡相关蛋白的表达及激活。通过比色法检测Caspase酶活性。结果显示过表达CXCR4的MSCs和过表达CXCR4的SPION标记的MSCs的侵袭和增殖能力增强，而CXCR4下调显著抑制骨肉瘤细胞的增殖，并诱导其凋亡，AMD3100联合雷公藤甲素处理抑制骨肉瘤细胞增殖及侵袭，诱导其凋亡，并抑制ERK、AKT、STAT3和NF-κB的活性。而且，CXCR4下调诱导的骨肉瘤细胞凋亡是Caspase依赖的。qRT-PCR检测结果显示CXCR4过表达促进MSCs侵袭相关基因的表达，包括MMP9、MMP2等。通过敲减CXCR4，过表达AKT及LY294002干预，检测CXCR4对AKT通路相关蛋白的调控作用。构建NF-κB启动子荧光素酶报告质粒，检测CXCR4下调后对NF-κB通路的影响。结果显示CXCR4下调通过抑制PI3K/Akt/NF-κB通路诱导骨肉瘤细胞凋亡。小鼠骨肉瘤模型的研究显示AMD3100联合雷公藤甲素处理能够显著抑制骨肉瘤的生长和肺转移。通过qRT-PCR检测骨肉瘤和邻近组织中miR-181a的相对水平。通过荧光素酶报告确定miR-181a的靶基因并进行验证。结果显示骨肉瘤中miR-181a的相对水平明显高于邻近组织，雷公藤甲素通过抑制miR-181a靶向PTEN基因抑制骨肉瘤细胞的生长和侵袭。本研究发现CXCR4与MSCs及骨肉瘤细胞的增殖、侵袭及凋亡调控密切相关， AMD3100联合雷公藤甲素处理能够显著抑制骨肉瘤的生长和肺转移，CXCR4对MSCs及骨肉瘤细胞的调控涉及PI3K/Akt/NF-κB信号通路，这些发现为骨肉瘤的治疗提供了新的思路和靶点。