靶向干细胞硼中子俘获治疗人脑胶质母细胞瘤

Boron neutron capture therapy, a new targeting radiotherapy, can selectively kill tumor cells sparing normal tissue, and it has gained heart-stirring effect clinically on human brain glioma in developed country in recent years. Glioma stem sells (GSCs) is strongly radioresistant, and killing GSCs has been the focus of tumor therapy. Our research confirmed that BPA, a boronated compound, could be uptaked enough by glioma cell lines, and significant effect was obtained for BNCT of targeting EGFR positive glioblastoma multiforme (GBM). But GSCs can only absorb little boron. Uptake of boronated dendrimer CD133, a specific antigen of GSCs, antibody bioconjugate is being confirmed in GSCs of primary culture and isolation with CD133+/CD133- and mice with GSCs intracranial xenografts. The effect and molecular biological mechanism of BNCT on killing GSCs and tumor cells are being investigated by the first In-Hospital Neutron Irradiator (IHNI-1) in the world. The aim of this research is enhancing uptake of boron and effect of BNCT using boronated CD133 antibody bioconjugate, and it makes the foundation for BNCT in enhancement of GBM therapeutic effect.

硼中子俘获疗法（BNCT）是新型的选择性杀伤肿瘤细胞而不损伤正常组织的靶向放疗方法,近年来发达国家BNCT治疗人脑胶质瘤取得了明显的疗效。胶质瘤干细胞（GSC）具有较强的耐放射性,杀伤GSC成为肿瘤治疗的焦点。本团队前期研究证实胶质瘤细胞系/株能摄取足量硼化合物,靶向EGFR阳性胶质母细胞瘤（GBM）BNCT取得了可喜的疗效,但GSC硼摄取量相对较少。本研究首先制备靶向GSC特异抗原CD133抗体的硼化偶联物，采用GBM原代培养和分选的CD133+/CD133-的GSC及其原位移植瘤裸鼠模型进行研究,证实该偶联物可有效提高GSC和肿瘤硼吸收量，然后使用世界首台医院中子照射器,探讨BNCT对GSC和载瘤动物治疗的有效性及其分子生物学机制。本研究旨在应用CD133抗体硼化偶联物靶向GSC，有效提高GSC硼吸收量和BNCT的疗效，为将来临床BNCT治疗GBM奠定坚实的基础。

硼中子俘获疗法（boron neutron capture therapy，BNCT）是一种选择性的二元放射疗法，其释放高能量的α粒子对缺氧细胞及未增殖期的细胞同样具有杀伤力。BNCT适用于脑肿瘤和浅表的皮肤肿瘤。本研究发现胶质瘤细胞和黑色素瘤细胞吸收硼原子(boron,10B)携带剂BPA的剂量能够满足BNCT对细胞内10B含量的要求，富含10B的细胞经医院中子照射器（In-Hospital Neutron Irradiator，IHNI-1）照射后生存率和增殖率与不含10B的细胞比均明显下降，G2/M期细胞比例增高，cyclin B1和CDK1蛋白表达降低，Cyt c、caspase-9和P21[WAF1]蛋白表达增加。脑胶质瘤干细胞（glioma stem cells,GSCs）具有更高的放射耐受性，被认为是放射治疗后肿瘤复发的根源。CD133是GSCs公认的标记物。本研究制备能够被CD133+ GSCs高吸收的连接有CD133单克隆抗体的PAMAM dendrimers，将含有多个硼原子的制剂BSH包裹于其中，形成纳米微粒PD-CD133/BSH。包裹效率和承载能力分别为 (73.8±6.7)% 和(1.67±0.12)%。CD133+ GSCs SU2和U87s 经12h对0.1 µM PD-CD133/BSH 吸收效率分别为（95.7±4.6）%和（91.8±7.6）%，硼含量分别为0.86±0.07μg/107细胞和0.83±0.02μg/107细胞，经IHNI-1照射后生存分数明显降低。PD-CD133/BSH在原位胶质瘤移植裸鼠脑内均有良好的靶向性， CD133+细胞对PD-CD133/BSH吸收量较高，合并颅内注射PD-CD133/BSH 12 h和静脉注射BSH 1 h，在CD133+ SU2 GSCs移植裸鼠肿瘤组织内硼含量达25.7±5.8μg/g，经IHNI-1照射后平均生存时间为61.8±11.4 d，明显长于对照组。以上结果表明制备的纳米微粒PD-CD133/BSH对CD133+ GSCs细胞有良好的靶向性，适合用于BNCT治疗脑胶质瘤。