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Gene therapy for spinal code protection

脊髓保护的基因治疗

基本信息

批准号：
14370400
负责人：
TABAYASHI Koichi
金额：
$ 3.01万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370400/
关键词：
spinal cord protection thoracic aneurysm gene therapy paraplegia ORP150 CAG promoter adenoviral vector lipofection eNOS 遺伝子導入虚血再潅流障害脊髄虚血精髄保護 spinal cord injury lipofection naked DNA denovival vector spinal code injury

项目摘要

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.

成功的主动脉手术后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)是一种新的缺氧/缺血诱导的内质网相关伴侣蛋白。有报道称，过表达ORP150的培养神经元对低氧应激具有抵抗力。我们推测，在脊髓过度表达氧调节蛋白150kD(ORP150)可以减少缺血后运动神经元的死亡。从大鼠脑组织RT-PCR产物中扩增出ORP150基因，将其插入CAG启动子驱动的表达载体中。用Western blotting方法检测ORP150的表达。虽然将ORP150或LacZ表达载体体内脂质体导入兔脊髓，但均未检测到转导基因。与假手术对照组相比，脂质体治疗后的神经学评分降低。利用克隆在粘粒中的环状腺病毒基因组和Cre-loxP重组系统构建了ORP150的重组腺病毒载体。然而，无法获得足够数量的腺病毒颗粒。由于CAG启动子是一种基于肌动蛋白的杂合启动子，在大多数细胞中具有很强的活性，过量表达ORP150被认为对293细胞具有毒性。虽然有两种不同的基因传递方法用于脊髓保护，但结果有限。这些发现表明，ORP150的过度表达对神经细胞也有潜在的毒性作用，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。