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

在人类遗传性疾病的治疗方面，已开展了建立酶和基因替换方法的研究，并为溶酶体酶缺乏症的选择性组织靶向寻找药物来源。1.保护性蛋白/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，对包括内皮素-1(ET-1)在内的一系列神经肽具有保护作用。Galactosialidsis(GS)是一种人类PPCA缺乏症，具有常染色体隐性遗传特征，同时伴有这些酶活性的降低和包括神经功能异常在内的多种临床表现。对尸检的GS脑进行了ET-1的组织化学分析，ET-1是Cath A的内源性底物之一。ET-1样免疫反应性…结论：1.在有GS的神经细胞中，有更多的细胞异常增加。ET-1前体蛋白和ET-B受体cDNAs的同时表达导致ET-1在GS成纤维细胞中的积聚。利用在两个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了PPCA基因的破坏。获得1个PPCA基因缺失等位基因的细胞系。根据微生物神经氨酸酶的X-射线结构对人类神经进行同源建模。人类神经缺乏症(唾液酸中毒)中发现的氨基酸替换的结构效应预测，一些替换所在的结构域可能有助于与PPCA分子的相互作用。桑德霍夫病是一种由溶酶体β-氨基己糖苷酶β亚单位基因缺陷引起的神经节苷脂增多症。在基因突变的小鼠中，用来阐明疾病的发病机制，并发现特异性地表达某些类型的趋化因子，与GM2-神经节苷脂在脑内的积累平行。这一现象被认为适用于开发新的细胞替代疗法。从合成的具有抑制血管细胞黏附分子(VCAM-1)诱导人脐静脉内皮细胞(HUVEC)表达血管细胞黏附分子(VCAM-1)的生物活性中间体中发现了新的诱导细胞凋亡的化合物。较少

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