超声介导新型脑靶向载基因纳米粒跨血脑屏障治疗中枢神经系统疾病的可行性研究

The existence of blood brain barrier (BBB) in central nervous system (CNS) greatly increases the difficulty in gene therapy of CNS related diseases, therefore how to find a gene therapeutic method which can overcome the systemic circulation, BBB and meanwhile has higher efficiency, safety and targeting into the brain tissues is becoming the pursuit of researchers. Ultrasound combined with microbubbles, as a new method for targeting BBB disruption, has been extensively studied and show the promising future. With the progress of biomaterials and nano-medicine, according to the theory of acoustic droplet vaporization (ADV) and specific modification of carrier, we plan to construct a new brain-targeted gene-loaded nanoparticles by combined use of phase-shift and acoustic characteristic of nanoparticles and biological targeting, histocompatibility and effective gene-loaded ability of liposomes; then through the research on BBB/C6 glioma model in vitro and tumor-bearing mice model under the sonication, we will focus on how to further enhance the gene transfer efficiency in CNS, in addition, make a deeper exploration on the process and mechanism of ultrasound-mediated gene transfer in CNS by means of molecular biology, morphology, imaging and other detection methods. The objective of this project is to prove the feasibility of gene therapy for glioma by the method of ultrasound/ nanoparticles promoting exogenous gene across BBB and provide new strategies and novel ideas for the treatment of other CNS diseases.

中枢神经系统(CNS)由于血脑屏障(BBB)的存在,极大增加了其相关疾病基因治疗的难度, 探索一种即能通过体循环、跨越BBB，又能有效、安全、靶向进入脑组织的载基因体系一直是业界追寻目标。超声介导微泡作为靶向开放BBB策略已初见成效。随着生物材料和纳米医学的进步，根据“声致相变”学说和对载体进行特定修饰，针对BBB，我们提出利用纳米粒液气相变优势、声学特性和脂质体生物靶向、组织相容和高效载基因特点构建一种新型脑靶向载基因纳米粒，使其在超声作用下以体外BBB/C6胶质瘤和载瘤小鼠模型为研究对象，采用分子生物学，形态学和影像学等多种方法，对如何提高CNS基因转染效率进行重点研究，并对超声介导基因在CNS内转染过程及相关机制做深入探索。本项目在证实超声/纳米粒促进基因跨BBB治疗胶质瘤的可行性的同时，为其它CNS疾病治疗提供新策略和新思路。

我们以前的实验已经证实聚焦超声联合载有DNA的微泡能够无创、可逆、局部的开放血脑屏障（BBB），并且能够靶向的促进外源性基因转入中枢神经系统，但是由于微泡基因搭载率低、缺乏主动靶向等原因，目前还未出现治疗作用的微泡联合超声治疗颅内肿瘤。在我们的研究中，我们首次采用高基因负载率和多肽介导的主动靶向的脂质体复合物来抑制肿瘤生长。首先，我们将微泡与载有shBirc5的NGR靶向微球连接起来，它展示了很好的细胞靶向和基因负载率。接着，我们证实了与对照组相比，聚焦超声联合该脂质复合体能够有更高的基因转染率。最后，我们在细胞实验和动物实验中分别通过凋亡实验、实时多聚酶链式反应（RT-PCR）、免疫印迹（WB）和肿瘤体积测定、生存时间评估等评价了shBirc5对靶基因的沉默效应。实验组展现了明显的治疗效应，但是聚焦超声组、脂质复合体组和超声联合无靶脂质复合体组与对照组相比都没有明显的治疗作用（P＜0.01）。最后，我们的研究说明了有靶脂质复合体联合超声是应用RNA干扰技术治疗肿瘤的一个充满前景的方式。