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Experimental Gene Therapy Utilizing a Skin Transplant

利用皮肤移植进行实验性基因治疗

基本信息

批准号：
01870103
负责人：
TAKEUCHI Toshiyuki
金额：
$ 5.95万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Most of peptide hormones are produced as a propeptide and converted to a biologically active peptide during their transport from the Golgi apparatus to secretory granules. The conversion from propeptide to biologically active peptide is a unique function of endocrine cells. A number of propeptide hormone cDNAs, including a human proinsulin cDNA, have been introduced into both endocrine and non-endocrine cells, and those expressed in endocrine cells were generally processed correctly, while others expressed in non-endocrine cells were secreted constitutively as non-cleaved propeptides. However, inability of non-endocrine cells to convert propeptides to biologically active peptides does not mean their inability to process propeptides to active peptides. Non-endocrine cells including fibroblasts, hepatocytes, and lymphocytes produce biologically inactive propeptides and convert them to bioactive peptides by cleaving a unique consensus sequence -Arg^<-4>-X^<-3>-Lys/Arg^<-2>-Arg^<-2>@*X^<+1 … More >-. Thus, we took proinsulin as a model propeptide for its expression in non-endocrine cells, and constructed a mutant proinsulin DNA where peptide structure was comprized of B and A chains linked to C peptide by a pair of tetrabasic residues in the following order : B chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A chain, while the native proinsulin structure was B chain-Arg-Arg-C peptide-Lys-Arg-A chain. Both mutant and native proinsulin were expressed in a monkey kidney derived cell line, COS-7 cells that possess only a constitutive secretory pathway and is thought not to process native proinsulin to mature insulin. When mutant insulin was expressed, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium. Moreover, mutant proinsulin was completely converted to mature one by co-expressing it with the subtilisin-like endoprotease, furin. The insulin produced in COS cells presented an identical biological activity to a synthetic human insulin by the capability of incorporating 3-O-[ ^3] methyl-D-glucose into adipocytes. We demonstrated that the mutated proinsulin with a pair of tetrabasic residues at the processing sites can be converted to fully bioactive insulin in a non-endocrine cell line, COS-7 cells. In the future this type of a mutant proinsulin DNA construct may be utilized for a hybrid type artificial islet or for gene therapy. Less

大多数肽类激素是以前肽的形式产生的，并在从高尔基体转运到分泌颗粒的过程中转化为具有生物活性的肽。由前肽转化为生物活性肽是内分泌细胞的独特功能。许多前肽激素cDNA，包括人胰岛素原cDNA，已被引入到内分泌和非内分泌细胞中，并且在内分泌细胞中表达的那些通常被正确加工，而在非内分泌细胞中表达的其他前肽组成性地分泌为非切割的前肽。然而，非内分泌细胞不能将前肽转化为生物活性肽并不意味着它们不能将前肽加工为活性肽。包括成纤维细胞、肝细胞和淋巴细胞的非内分泌细胞产生生物学上无活性的前肽，并通过切割独特的共有序列-Arg^<-4>-X^<-3>-Lys/Arg^<-2>-Arg^<-2>@*X^<+1将它们转化为生物活性肽 ...更多信息 >-.因此，我们以胰岛素原作为在非内分泌细胞中表达的模型前肽，构建了一个突变的胰岛素原DNA，其中肽结构是由B链和A链通过一对四碱基残基连接到C肽，顺序如下：B链-Arg-Arg-Lys-Arg-C肽-Arg-Arg-Lys-Arg-A链，而天然胰岛素原结构是B链-Arg-Arg-C肽-Lys-Arg-A链。突变体和天然胰岛素原均在猴肾衍生细胞系COS-7细胞中表达，COS-7细胞仅具有组成型分泌途径，并且被认为不将天然胰岛素原加工成成熟胰岛素。当突变体胰岛素表达时，约60%的总免疫反应性胰岛素在培养基中以成熟胰岛素的形式出现。此外，突变胰岛素原完全转化为成熟的共表达它与枯草杆菌蛋白酶样内切蛋白酶，弗林蛋白酶。COS细胞中产生的胰岛素通过将3-O-[ ^3]甲基-D-葡萄糖掺入脂肪细胞的能力而呈现与合成人胰岛素相同的生物活性。我们证明了在加工位点具有一对四碱基残基的突变胰岛素原可以在非内分泌细胞系COS-7细胞中转化为完全生物活性的胰岛素。将来，这种类型的突变胰岛素原DNA构建体可用于杂交型人工胰岛或基因治疗。少