Gene therapy for spinal code protection

主动脉手术成功后的脊髓损伤是患者无法接受的并发症，需要新的脊髓保护方法。氧调节蛋白150kD （ORP150）是缺氧/缺血诱导的一种新型内质网相关伴侣蛋白。据报道，培养的过表达ORP150的神经元对低氧应激具有抗性。我们假设脊髓中氧调节蛋白150kD（ORP150）的过度表达会减少缺血后运动神经元的死亡。从大鼠脑RT-PCR产物中获得ORP150 cDNA，并将其插入CAG启动子驱动的表达质粒中。Western blotting法证实ORP150的表达。通过对兔脊髓进行ORP150或LacZ表达质粒体内脂质转染，均未检测到转导基因。与假对照组相比，抽脂后神经学评分降低。利用克隆的环状腺病毒基因组和Cre-loxP重组体系构建表达ORP150的腺病毒载体。然而，未能获得足够数量的腺病毒颗粒。CAG启动子是一种基于肌动蛋白的杂交启动子，在大多数细胞中具有很强的活性，ORP150的过量表达被认为对293细胞具有毒性。虽然计划了两种不同的基因传递方法来保护脊柱，但结果有限。这些发现表明，ORP150的过度表达也可能对神经细胞有毒性，当我们将这种方法应用于主动脉手术时，控制ORP150的表达水平被认为是重要的。

Spinal cord injury after successful aortic surgical operations is an unacceptable complication for patients, and new spinal cord protection method is required. Oxygen-regulated protein 150kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. And it was reported that cultured neurons overexpressing ORP150 were resistant to hypoxemic stress.We hypothesized that overexpression of Oxygen-regulated protein 150kD(ORP150) in spinal cord would reduced motor neuron death after ischemia. ORP150 cDNA was obtained from RT-PCR products of Rat brain, and was inserted into expressing plasmid driven by CAG promoter. Expression of ORP150 was confirmed by using Western blotting method. Though in vivo lipofection of ORP150 or LacZ expression plasmid were tried to rabbit spinal cord, transduced gene were not detected at all. Neurological score after lipofecton reduced as compared to sham control. ORP150 expressing adenovirus vector was constructed by using circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system. However, enough amount of adenoviral particles could not be obtained. As CAG promoter, an actin-based hybrid promoter, has very strong activities in most cells, excess expression of ORP150 was considered to have toxicity against 293 cells. Though two different gene delivery methods were planed for spinal protection, results were limited. These findings suggested that excess expression of ORP150 potentially also had toxicity against nerve cells, and to manage the expression levels of ORP150 is considered to be important when we apply this method to aortic surgery.