结合p53C末端的双靶向多功能含硼脂质体的构建及对神经胶质瘤中子照射协同治疗的研究

Glioma is the most common intracranial tumor at present. Because of the invasive growth of tumor cells and insensitivity to chemotherapy and radiotherapy, glioma has a high relapse frequency and is still one of the tumor with poor prognosis after surgical operation. Boron neutron capture therapy (BNCT) is a binary method for the treatment of cancer based on tumor-selective 10B delivery followed by radiation with low energy thermal neutrons. BNCT provides a way to selectively destroy malignant cells and spare normal cells even tumor cells have spread into normal tissues. The world's first hospital neutron irradiator has been built in China, but clinical research has not been carried out yet. Based on successful prokaryotic expression of glioma targeting peptide fusion protein, we aim at designing and expressing a multifunctional targeting therapy protein fused with glioma targeting peptide, fusogenic peptide, cell-penetrating peptide (CPP) and tumor p53 C terminus. The fusion protein and Angiopep-2 targeting blood brain barrier (BBB) are coconjugated on the surface of PEG liposome encapsulating BSH, resulting in the multifunctional liposome. The prepared liposomes are supposed to pass through the BBB and blood-tumor barrier (BTB) and delivered BSH into glioma cells. The fusogenic peptide, HA-2, help the release of endocysed liposome containing BSH and therapy peptide P53 C terminus into cytoplasm. Thus the glioma maybe eliminated through promoting the apoptosis of tumor cells and low dose neutron bombardment at the same time. The p53 C terminus combined therapy in BNCT may avoid the injury of normal tissue in the eradication of tumor tissue. This research will provide theoretical data for BNCT in basic research in China and an effective way for the treatment of glioma and other tumors.

神经胶质瘤是最常见的颅内肿瘤，由于肿瘤细胞的侵袭生长及对化疗放疗的不敏感导致了高复发性。硼中子俘获治疗（BNCT）是一种选择性杀伤肿瘤细胞的治疗方法，其成功前提是将含硼化合物高效靶向地导入肿瘤细胞中。我国已经建成世界首台医院中子照射器，但临床研究尚未开展。本课题组在成功表达神经胶质瘤靶向肽融合蛋白的研究基础上，拟将肿瘤靶向肽与p53 C末端、促溶肽和细胞穿膜肽一起设计成小分子量的多功能靶向治疗蛋白，与透过血脑屏障（BBB）的Angiopep-2靶向肽连接到含硼脂质体上，构建的多功能含硼靶向脂质体能够通过BBB和肿瘤血管屏障，将脂质体中的硼药物传送到脑肿瘤细胞中，并利用促溶肽使这种多功能脂质体的P53C和含硼药物快速释放到细胞质中，在促进肿瘤细胞凋亡的同时，结合较小剂量的中子照射消灭肿瘤组织。本研究将为结合蛋白治疗的BNCT基础研究提供理论依据，为神经胶质瘤以及其他肿瘤的治疗提供有效的手段。

硼中子俘获疗法(Boron Neutron Capture Therapy, BNCT)是一种双元的选择性杀伤肿瘤细胞的治疗方法，其成功前提是将含硼化合物高效靶向地导入肿瘤细胞中。尽管我国世界首台用于治疗恶性肿瘤的医院中子照射器（IHNI）处于领先水平，但硼药物和临床研究相对滞后。本研究将多功能靶向治疗蛋白与硼脂质体载体结合，为BNCT提供新型的组合治疗载体。. 我们首先构建了神经胶质瘤靶向肽（GL1）融合蛋白并检测了其对神经胶质瘤的靶向能力，连接穿膜肽TAT的融合蛋白GL1-EGFP-TAT效果优于GL1-EGFP。为了增加蛋白的表达量，引入ZZ作为融合蛋白并加入蛋白酶切位点，将p53 C与MDM2分别与GL1、riHA2（促溶肽）和TAT的基因序列相融合，在大肠杆菌中表达GHPc+mT等四种多功能融合蛋白。四种融合蛋白对细胞增殖的抑制效果呈剂量和时间依赖性，GHPc+mT效果最为明显，对U87△EGFR的抑制率约为52.6%。流式细胞仪检测结果表明：融合蛋白GHPc+mT能诱导U87△EGFR和paU87细胞早期凋亡。Western blotting 分析表明GHPc+mT促进细胞凋亡的能力最强。克隆形成实验和Transwell分析表明GHPc+mT对神经胶质瘤细胞的克隆形成和迁移、侵袭能力具有显著的抑制作用。. 我们也在大肠杆菌中高效表达了靶向肽融合蛋白Angiopep-2-EGFP（ANG-E）和CTX-EGFP，ANG-E对人原代神经胶质细胞不具有靶向性，ANG-E和CTX-EGFP对不同神经胶质瘤细胞均具有靶向能力。ICP-AES测试结果表明：ANG-E修饰的脂质体硼载体能够将10B有效地导入到不同的神经胶质瘤细胞中。荷瘤裸鼠体内实验表明：ANG-E修饰的脂质体硼载体的裸鼠体内硼导入效率高于没有修饰的脂质体硼载体。. 中子照射三种含硼肿瘤细胞后，增殖能力均被显著抑制。流式细胞术检测出肿瘤细胞发生早期凋亡。基因芯片分析结果表明：BNCT组细胞中包括TP53和CDKN2A在内的基因表达发生显著改变。Western blotting分析表明，BNCT能够通过上调P53表达调控Bcl-2/Bax线粒体凋亡和P16参与的细胞周期信号通路。.本课题共发表了研究论文7篇， 其中Sci论文6篇。培养硕士研究生7名。参加国际和国内BNCT会议各一次。