基于MFH技术的CD44-shRNA/DDP纳米脂质体靶向治疗卵巢癌的研究

Mn0.5Zn0.5Fe2O4 nanoparticles, novel functional magnetic nano-materials, will be prepared in this project.As gene transfer carriers and magnetic induction mediums for thermotherapy, they will be combined with CD44-shRNA and DDP to prepare CD44-shRNA/DDP magnetic nanoliposomes. After being injected to ovarian cancer tissue, the nanoliposomes will be heated by magnetic induction directionally. While heat killing tumor, DDP encapsulated in the nanoliposomes can release to kill cancer.Meanwhile, CD44-shRNA plasmids can get into cancer cells to block CD44 gene expression. As a result, a multiple targeting killing of siRNA, DDP and hyperpyrexia will may be realized. In research,some related characteristics of the nanolipisomes will be examined. In order to explore whether the combination therapy can effectively inhibit ovarian cancer development, metastasis and recurrence from the whole, tissue, cell and molecular level, their in vitro and in vivo curative effects on ovarian cancer will be detected;some side-effects will be evaluated;the invasion, migration and tumorigenesis abilities of ovarian cancer cells will be tested after treatment;CD44,VEGF and survivin genes expression will be analyzed.The characteristic and innovation of this project lies in the organic combination of chemotherapy,gene therapy and thermotherapy to take a complementary and synergistic effect by using magnetic nanomaterials as carriers.This may become a new effective combination therapy to treat cancer.

本项目拟采用Mn0.5Zn0.5Fe2O4纳米粒作为磁热疗介质和基因治疗载体，与CD44-shRNA真核表达质粒和顺铂（DDP）结合，制备CD44-shRNA/DDP磁性纳米脂质体，介入至卵巢癌组织，体外磁感应定向加热，一方面热疗和顺铂可杀伤肿瘤细胞，同时CD44-shRNA质粒进入肿瘤细胞内使CD44基因表达沉默，实现“基因、顺铂、高热”对卵巢癌的靶向联合杀伤作用。检测磁性纳米脂质体的相关生物学特性，以对卵巢癌细胞的增殖抑制和凋亡诱导作用及体内抑瘤率为指标，检测其对卵巢癌的治疗作用，同时观察毒副作用，分析治疗后卵巢癌细胞侵袭、迁移和成瘤能力变化以及CD44、VEGF、survivin等基因表达改变，从动物、组织、细胞和分子水平上评价联合治疗能否有效抑制卵巢癌发展、转移和复发。本研究的特色是以磁性纳米材料为载体，使基因治疗、化疗、热疗有机结合，优势互补，有可能形成一种新的肿瘤联合治疗方法。

卵巢癌严重威胁着广大妇女的健康，死亡率占妇科肿瘤的首位。本项目采用锰锌铁氧体纳米粒（MZF-NPs）作为磁热疗介质和基因治疗载体，与CD44-shRNA质粒和顺铂（DDP）结合，制备CD44-shRNA/DDP磁性纳米脂质体，输入卵巢癌组织，体外磁感应加热，一方面热疗和顺铂可杀伤肿瘤细胞，同时CD44-shRNA质粒进入肿瘤细胞内使CD44基因表达沉默，从而将“CD44基因封闭、顺铂化疗、磁流体热疗”有机结合对卵巢癌进行靶向联合治疗研究。结果显示，成功构建了CD44-shRNA/DDP磁性纳米脂质体；PEG修饰的MZF-NPs可以有效地结合、保护和释放CD44-shRNA质粒，具有较高的转染效率，显示了良好的基因转运潜能；PEG-MZF/DDP复合纳米粒具有较好的升温控温效果，在外加交变磁场作用下，温度可以稳定在43℃左右，适合用于肿瘤热疗；CD44-shRNA/DDP纳米脂质体磁感应加热对卵巢癌HO8910细胞具有明显的增殖抑制和侵袭抑制作用，并诱导其凋亡，在体内显著抑制裸鼠卵巢癌移植瘤的生长，效果远远优于单纯的DDP、shRNA、MFH、DDP/MFH，具有较好的互补协同效应，显示了联合治疗的明显优势；基因检测结果提示，CD44-shRNA/DDP纳米脂质体磁感应加热可能通过下调凋亡抑制基因survivin、bcl-2、bcl-xl蛋白表达和上调促凋亡基因Caspase-3、Caspase-9蛋白表达，诱导肿瘤细胞凋亡，同时通过抑制VEGF、CD44蛋白表达，抑制细胞侵袭及抗肿瘤新生血管形成，从而抑制卵巢癌的生长、侵袭迁移。病理组织学、生化、血细胞等检测显示，DDP/CD44-shRNA/MFH联合治疗未见明显毒副作用。本项目研究成果为卵巢癌临床治疗提供了新的思路和实验依据。