超声微泡造影剂介导siRNA基因沉默与诱导双向组合式基因治疗椎间盘退变的实验研究

Intervertebral disc degeneration (IDD) is induced by multiple key factors, which mainly include upregulation of cell apoptosis, hyperactivity of MMPs (e.g., MMP-3), as well as downregulation of molecules that are responsible for synthesis of extracellular matrix (ECM) in the nucleus pulposus (NP). We hypothesized here that using gene therapy-mediated combined approaches targeting these factors would provide an efficient preventive or therapeutic strategy against development or progression of IDD. In this study, three different vectors including Fas-siRNA and MMP3-siRNA plasmid vectors, as well as adeno-associated virus vector harboring SOX-9 (AAV-SOX9) will be generated. These vectors, either alone or combined, will be transferred both in vitro and in vivo using newly developed ultrasound microbubble contrast media mediated gene transfer technique. For in vitro studies, vectors will be introduced into human early degenerative NP cells. For in vivo studies, These vectors will be transferred into lumbar discs in Rhesus monkeys undergoing early IDD, a model that have been induced by a minimally invasive approach. Following a series histopathological and biochemical analyses, we aim at investigating whether combined gene therapy would prove to be synergistically effective in simutaneously abrogating cell apoptosis and ECM degradation, while stimulating ECM production in NP.

椎间盘退变是多因素协同作用不断进展的过程，关键性环节包括：髓核细胞过度凋亡，基质金属蛋白酶(如MMP-3）活性增高，促进合成细胞外基质正向调节因子（如SOX-9）减少。这些综合因素最终导致髓核细胞外基质合成减少，髓核承受压力能力降低，诱导并加重退变。如能采取措施阻断这些因素，就可从根本上延缓椎间盘退变。基于这一假说，本研究在前期工作基础上，构建荧光标记FAS-siRNA、MMP3-siRNA质粒表达载体和腺相关病毒介导SOX-9载体。建立椎间盘退变细胞和动物模型基础，应用新型超声微泡造影剂配合超声辐射技术，将Fas-siRNA、MMP3-siRNA及AAV-SOX9分别或联合导入早期退变髓核细胞，通过细胞学与动物实验尝试应用基因沉默与基因诱导双向组合式基因治疗模式，在抑制髓核细胞凋亡及阻断细胞外基质降解基础上，促进髓核细胞外基质分泌，以期产生协同生物学效应，从而有效延缓或逆转椎间盘退变。