声穿孔联合携NET-1siRNA双功靶向纳米微泡在肝癌治疗中的作用及其机制研究

Gene therapy has become a new choice for patients with liver cancer.However，the key to gene therapy is how gene is transfected efficiently and safely.The study showed that NET-1 is expressed highly in liver cancer tissue.It has been implicated in signal transduction,cell proliferation and differentiation. Our previous study found that NET-1siRNA transfected by ultrasound combined with microbubble can effectively inhibit the proliferation of liver cancer cells.But,the efficiency of transfection is limited. Then, we hypothesize that ultrasound combined with targeting nano microbubble carried with NET-1siRNA is applied in the treatment of liver cancer. To test this hypothesis, we will use G-PLL found in our before study as a match for the target nano microbubble, and the NET-1 siRNA will be loaded in this bubble which is only nanoscale size. In the ultrasonic irradiation,the compound can go into the tumor capillaries, and then across the high permeability blood vessel wall into tumor cells, which can effectively solve the problems of siRNA easily degradation in the body and difficultly entering the targeting cell.At the same time, this study will furtherly research NET-1 siRNA treatment mechanism of liver cancer. This research has targeted the carriage,targeted release, targeting convergence of the triple function. Ultrasound combined with nano microbubble-mediated gene transfection is to lay a foundation for revealing the mechanism of NET-1 affectting liver cancer.It will provide a new idea for the treatment of liver cancer.

基因治疗已经成为肝癌患者的新选择，然而困扰基因治疗的关键是如何高效、安全高效转染基因。研究表明，NET-1基因在肝癌组织中高表达，参与信号转导、细胞增殖和分化。我们前期研究发现超声联合微泡转染NET-1siRNA能够有效抑制肝癌细胞的生长，但基因转染效率有限。据此提出假设，应用超声联合靶向载NET-1siRNA纳米微泡治疗肝癌，将前期研究发现的G-PLL作为配体制备靶向性纳米微泡，并将NET-1siRNA包裹入其中构成仅有纳米级大小的载基因复合物。该复合物在超声波的照射下经过通透性增大的血管壁进入肿瘤细胞内，有效地解决siRNA在体内易降解难以进入靶细胞这一难题。本课题将通过此种方法进一步研究NET-1siRNA治疗肝癌的作用机制。本研究将从超声介导靶向载基因纳米微泡入手，实现NET-1siRNA的靶向运载、靶向释放、靶向汇聚，为肝癌治疗提供新思路。

NET-1基因在肝癌组织中高表达，参与信号转导、细胞增殖和分化。本项目在前期工作基础上，应用低频超声联合靶向载NET-1siRNA纳米微泡治疗肝癌，将前期研究发现的G-PLL作为配体制备靶向性纳米微泡，并将NET-1siRNA包裹入其中构成仅有纳米级大小的载基因复合物。该复合物在低频超声的辐照下，经过通透性增大的细胞膜进入肿瘤细胞内，有效地解决非病毒siRNA载体转染率较低这一难题。首先以磷脂复合物为原料、以半乳糖苷化多聚赖氨酸为靶向配体，利用薄膜水化法配合机械振荡法制备靶向纳米微泡，利用生物素-亲和素系统，将NET-1 siRNA与靶向纳米微泡偶联，制备成载NET-1siRNA肝癌靶向纳米微泡。进而采用CGZZ型低频超声基因转染治疗仪分别进行细胞实验与动物实验。采用人缘肝癌细胞系SMMC-7721，动物实验建模选用用Balb/c免疫缺陷鼠。在细胞学实验中， Quantitative Real-time PCR及Western Blot实验均证实通过该转染方法，NET-1 mRNA与NET-1蛋白被显著抑制。通过transwell实验证实治疗组细胞迁移能力和侵袭能力明显低于阴性对照组。通过流式细胞仪检测治疗组转染率高达73.2%。在动物性实验中，首先对转染后的各组动物进行活体多光谱成像，实验证实载基因靶向纳米微泡具备良好的体内寻靶能力。之后观察并记录肿瘤大小及生存期，治疗组肿瘤生长明显减慢并逐渐缩小，生存期也较对照组延长。处死裸鼠进行免疫组化检查显示活体动物实验中，治疗组荷瘤鼠NET-1蛋白的表达明显降低，各脏器均未见明显损伤。基于上述研究成果课题组得出结论，载基因靶向纳米微泡联合低频超声可以高效的转染基因。课题组成员还探索使用更高效的靶向配体磷脂酰肌醇蛋白聚糖3（GPC3），制备出具备更强靶向能力的纳米微泡，进一步提高转染率。项目资助发表SCI收录论文3篇，另有投稿外审中3篇。资助发表国家核心期刊论文4篇，申请国家发明专利两项，培养硕士生2名，其中1名已经取得硕士学位，1名在读。项目经费各项支出基本与预算相符，剩余经费用于后续文章发表。