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

在人类遗传性疾病的治疗中，人们一直在研究建立酶和基因替代方法，并为受溶酶体酶缺陷影响的选择性组织靶向寻找药物资源。在项目实施过程中，取得了以下新的研究成果：1.保护蛋白/组织蛋白酶A（PPCA）是一种多功能糖蛋白，其对溶酶体神经氨酸酶（Neur）和β-半乳糖苷酶（β-Gal）表现出保护作用，并且对包括内皮素-1（ET-1）在内的神经肽子集表现出丝氨酸羧肽酶（组织蛋白酶A ; Cath A）活性。半乳糖唾液酸沉积症（Galactosialidosis，GS）是一种具有常染色体隐性遗传特征的人类PPCA缺陷症，伴随着这些酶活性的同时降低和包括神经系统异常在内的多种临床表现。组织化学分析的尸检GS脑对ET-1，一个假定的内源性底物的Cath A，进行。ET-1样免疫反应性 ...更多信息 结果：1. GS处理的神经细胞中，GSH-Px活性异常增高.同时表达ET-1前体蛋白和ET-B受体cDNA可引起GS成纤维细胞系中ET-1的积聚.使用在两个loxP序列之间含有耐药基因盒的靶向载体在人星形细胞瘤细胞系中进行PPCA基因破坏。获得了PPCA基因一个等位基因缺失的细胞系.基于微生物神经氨酸苷酶的X射线结构进行了人神经元的同源性建模。在人类Neur缺陷症（唾液酸沉积症）中鉴定的氨基酸取代的结构效应预测了某些取代所在的结构域可能有助于与PPCA分子的相互作用. Sandhoff病是由溶酶体β-氨基己糖苷酶β-亚基的遗传缺陷引起的GM 2-神经节苷脂沉积症。在基因破坏的小鼠作为疾病模型被用来阐明发病机制，并发现特异性表达的某些类型的趋化因子平行于GM 2-神经节苷脂在脑中的积累。这种现象被认为适用于开发新的细胞替代疗法.从生物活性盐咯啉（halichroline）的合成关键中间体中发现了新的诱导凋亡的化合物，盐咯啉对人脐血管内皮细胞（HUVEC）上的血管细胞粘附分子（VCAM-1）的诱导表达具有抑制作用。少

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