Development and application of tissue-directed medicinal resources based on biotechnology

基于生物技术的组织定向药用资源开发与应用

基本信息

批准号：
12470503
负责人：
ITOH Kohji
金额：
$ 9.09万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

For therapy of human genetic diseases, the research had been performed to establish the enzyme and gene replacement methods and to discover the medicinal resources for the selective tissue targeting affected with lysosomal enzyme deficiencies. During the term of project, new findings were obtained as follows :1. Protective protein/cathepsin A (PPCA) is a multifunctional glycoprotein that exhibits the protective effects on lysosomal neuraminidase (Neur) and β-galactosidase (β-Gal), and serine carboxypeptidase (cathepsin A ; Cath A) activity on a subset of neuropeptides including endothelin-1 (ET-1). Galactosialidosis (GS) is a human PPCA deficiency with autosomal recessive genetic trait, accompanied by the simultaneous decrease of these enzyme activities and multiple clinical manifestations including neurological abnormalities. Histochemical analysis of the autopsied GS brain against ET-1, one of the putative endogenous substrates of the Cath A, was performed. ET-1-like immunoreactivity … More in the neural cells with GS was demonstrated to increase abnormally.2. Simultaneous expression of ET-1 precursor protein and ET-B receptor cDNAs caused the accumulation of ET-1 in the GS fibroblastic cell line.3. PPCA gene disruption in a human astrocytoma cell line was performed using the targeting vector containing the drug-resistant gene cassette between the two loxP sequences. The cell line with one disrupted allele of PPCA gene was obtained.4. Homology modeling of human Neur was performed on the basis of X-ray structure of microbial neuraminidases. Structural effects of amino acid substitutions identified in human Neur deficiency (sialidosis) predicted that the domain in which some substitutions locate might contribute to the interaction with PPCA molecule.5. Sandhoff disease is a GM2-gangliosidoses caused by the genetic defect of the β-subunit of lysosomal β-hexosaminidase. In the gene-disrupted mice as the disease model were used to elucidate the pathogenesis and were found to express specifically some types of chemokine parallel to the accumulation of GM2-ganglioside in the brain. This phenomenon was considered to be applicable to develop novel cell replacement therapy.6. Novel apoptosis-inducing compounds were discovered from the synthetic key intermediates of the bioactive halichroline that has the inhibitory action on the induced-expression of vascular cell adhesion molecule (VCAM-1) on the human umbilical vascular endothelial cells (HUVEC). Less

在人类遗传疾病的治疗方面，我们进行了研究，建立酶和基因替代方法，并发现针对溶酶体酶缺陷的选择性组织的药用资源。项目期间，获得了以下新发现： 1. 保护蛋白/组织蛋白酶 A (PPCA) 是一种多功能糖蛋白，对溶酶体神经氨酸酶 (Neur) 和 β-半乳糖苷酶 (β-Gal) 具有保护作用，半乳糖唾液酸沉积症 (GS) 等神经肽子集上的丝氨酸羧肽酶（组织蛋白酶 A；Cath A）活性是一种具有常染色体隐性遗传特征的人类 PPCA 缺陷，伴随着这些酶活性的同时降低。以及多种临床表现，包括针对 ET-1（Cath A 的假定内源性底物之一）尸检 GS 脑的组织化学分析。 GS 神经细胞中的 ET-1 样免疫反应性异常增加。 2． 3.使用含有两个loxP序列之间的耐药基因盒的靶向载体对人星形细胞瘤细胞系进行PPCA基因破坏，该细胞系具有一个被破坏的等位基因。 4.根据微生物神经氨酸酶的X射线结构对人类Neur进行同源建模，预测了人类Neur缺陷（唾液酸中毒）中氨基酸取代的结构域。可能与PPCA分子相互作用有关。5．Sandhoff病是一种由溶酶体β亚基基因缺陷引起的GM2神经节苷脂病。 β-氨基己糖苷酶在作为疾病模型的基因被破坏的小鼠中被用来阐明其发病机制，并发现其特异地表达某些类型的趋化因子，与GM2-神经节苷脂在大脑中的积累平行。开发新型细胞替代疗法。6．从具有生物活性的卤代盐关键中间体中发现了新型凋亡诱导化合物，该化合物对血管细胞粘附分子的诱导表达具有抑制作用。 (VCAM-1) 对人脐血管内皮细胞 (HUVEC) 的影响较小。