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.

成功主动脉手术手术后，脊髓损伤是患者不可接受的并发症，需要新的脊髓保护方法。氧气调节的蛋白150KD（ORP150）是一种新型的内质 - 逆转蛋白质相关的伴侣，由低氧/缺血诱导。据报道，过表达ORP150的培养神经元对低氧胁迫有抵抗力。我们假设脊髓中氧气调节的蛋白150KD（ORP150）的过表达会降低缺血后运动神经元死亡。 ORP150 cDNA是从大鼠脑的RT-PCR产物中获得的，并插入由CAG启动子驱动的质粒中。使用蛋白质印迹法证实了ORP150的表达。尽管在兔脊髓中尝试了ORP150或LACZ表达质粒的体内lipofection，但根本没有检测到转导的基因。与假对照相比，LipoFecton后的神经学评分降低。 ORP150表达腺病毒载体是通过使用克隆在cosmid和Cre-loxp重组系统中的腺病毒基因组的圆形形式构建的。但是，无法获得足够数量的腺病毒颗粒。由于基于肌动蛋白的杂种启动子CAG启动子在大多数细胞中具有非常强大的活性，因此ORP150的过量表达被认为对293个细胞具有毒性。尽管计划了两种不同的基因输送方法进行脊柱保护，但结果受到限制。这些发现表明，ORP150的过量表达可能也可能对神经细胞有毒性，并且当我们将这种方法应用于主动脉手术时，管理ORP150的表达水平被认为很重要。