外泌体靶向运输TRP-PK1多肽抑制喉癌的作用及其机制研究

Laryngeal cancer is one of the most common malignant tumour in head and neck tumor, the incidence is only lower than lung cancer in respiratory system. The main treatment of laryngeal cancer is surgery, and the patient will loss of vocal organs after total laryngectomy and with poor quality of life. This highlights the need for further research and innovation in the new target therapy of laryngeal cancer. The researches showed that the cell membrane potential and followed calcium signal played important role in proliferation, migration and apoptosis of laryngeal carcinoma cells. Our research group independent designed and synthesised the TRP-PK1 peptides which might self-assembly potassium channel and mediate potassium efflux. TRP-PK1 peptides induced hyperpolarization and declined the proliferation and migration, enhanced the apoptosis. Nuclear magnetic resonance has unique advantages in the study of molecular interactions and molecular structure in the solution, while magnetic resonance imaging is superior in soft tissue contrast and spatial resolution, and can be used for three-dimensional depth detection. In order to reveal the mechanism of targeted delivery for TRP-PK1 peptides in vivo and the role of inhibition to laryngeal cancer, we plan to do the followed researches in cell and animal level through liquid nuclear magnetic resonance titration, paramagnetic relaxation enhancement method and high field magnetic resonance imaging: (1) the dynamic process of TRP-PK1 peptides and cell membrane and the structure principle; (2) to set up exosomes that may delivery TRP-PK1 peptides targeting to laryngeal carcinoma by specific identification and combination of RGD sequence and integrin ɑvβ3 receptor; (3) the inhibition and specificity of TRP-PK1 peptides to laryngeal cancer through targeted delivery. This study will be helpful to explore new delivery methods of targeted drug and targeted drugs to inhibit laryngeal cancer, and provide basic data for targeted therapy to laryngeal cancer.

喉癌发病率逐年升高，但治疗手段有限，急需有效的靶向治疗方法和药物。申请人前期研究发现TRP-PK1多肽可在喉癌细胞膜形成钾离子通道，引起细胞膜超极化和胞内钙浓度升高，从而降低喉癌细胞增殖、迁移并促进凋亡。核磁共振在研究溶液中分子相互作用和分子结构方面具有独特的优势，而磁共振成像则具有软组织对比度和空间分辨率高、可进行三维深度检测等特点。为探索在体靶向运输TRP-PK1多肽与其抑制喉癌的作用，本项目拟通过液体核磁共振滴定法、顺磁弛豫增强法和高场强磁共振成像研究以下内容：（1）TRP-PK1多肽在细胞膜形成钾离子通透通道的动态过程与结构原理；（2）通过RGD序列-整合素ɑvβ3受体的特异性识别，建立在体靶向喉癌运输TRP-PK1多肽的外泌体；（3）外泌体靶向运输TRP-PK1多肽对喉癌的抑制作用及其特异性。该研究将有助于探索新型的靶向输送药物方法和靶向抑制喉癌药物，为喉癌靶向治疗提供基础数据。

喉癌是头颈部常见的恶性肿瘤之一，治疗方式主要以手术切除为主，靶向治疗将有效避免手术切除对生活质量的严重影响并可提高预后效果。本研究利用可以插入细胞膜中形成K+通道的TRP-PK1 多肽构建了TRP-PK1-iRGD-外泌体的靶向运输系统，体内外实验证明具有良好的靶向性。用于靶向运载化疗药物洛铂治疗喉癌，既可取得良好效果，又减小了化疗药物引起的体重减轻、肝脏损伤等毒副作用。同时证明TRP-PK1本身也具有抑制肿瘤细胞增殖和迁移的作用。本研究也构建超小型氧化铁纳米粒子造影剂的体系，与TRP-PK1-iRGD-外泌体的靶向运输系统相结合，可以作为有效的强磁场造影剂指示肿瘤大小，并成为诊疗一体化的体系。通过TRP-PK1目标蛋白载体构建与表达，利用液态核磁共振技术检测micelle状态下的TRP-PK1小肽的1H-15N HSQC谱图。另外，我们也观察了不同强度的磁场对喉癌细胞表型和外泌体分泌的影响，发现10T超导磁场环境下培养头颈癌细胞株HN4 24h促进肿瘤细胞焦亡、抑制肿瘤细胞增殖和迁移，但可促进外泌体分泌。 在本课题的研究过程中使用稳态强磁场实验装置（SHMFF）所属的SM3 850M 液相核磁共振谱仪和超导磁体及配套生物样品培养系统共168小时。通过本课题的研究，发表SCI论文8篇，待发表1篇；国际学术会议摘要1篇；研究生大论文3篇。国际会议墙报1人次。培养硕士研究生5名，已毕业3名，待毕业答辩2名。