权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Fundamental study of gene therapy for epidermolysis bullosa : Development of gene therapy targeting keratinocyte

大疱性表皮松解症基因治疗的基础研究：针对角质形成细胞的基因治疗的开发

基本信息

批准号：
08670941
负责人：
SAWAMURA Daisuke
金额：
$ 1.41万
依托单位：
HIROSAKI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

The dystrophic form of epidermolysis bullosa (DEB) is characterized by subliminal densa blister, and mutations in the type VII collagen gene have been shown to underlie DEB. To address gene therapy of DEB, we tried to introduce type VII collagen gene to keratinocytes. We first cloned a 9-kb full-length cDNA of the type VII collagen gene, constructed a expression vector of the cDNA and introduced it to rat kerationcytes using the naked DNA method. The results demonstrated that the expression of transgene was found in kerationcytes 24 h later and in basement membrane zone a week later. Success in expression of this collagen suggested a possibility of gene therapy for DEB.Also, we constructed an IL-6 expression vector and introduced it to rat keratinocytes. Introduction of the IL-6 gene induced erythema macroscopically, and epidermal hypertrophy and lymphocytic infiltration microscopically. Next, we could show that pre-introduction of IL-6 antagonsit genes to keratinocytes inhibited inflammation by post-introduction of the IL-6 gene. Since IL-6 production was increased in keratinocytes of psoriasis, our result indicated possibility of gene therapy for psoriasis. In keratinocytes of psoriasis. Furthermore, we introduced IL-10 gene to keratinocytes and then measured serum level of IL-10. Significant level of IL-10 was found in serum and circulating IL-10 inhibited contact hypersensitivity at distant areas of the skin. This result suggested gene therapy for systemic diseases using keratinocyte as a bioreactor.Besides the above results, we develop new gene transfer methods to keratinocytes and found several mutations in patients with DEB.

营养不良型大疱性表皮松解症(DEB)的特点是以阈值下致密水泡为特征，而VII型胶原基因的突变被证明是DEB的基础。为了解决DEB的基因治疗问题，我们尝试将VII型胶原基因导入角质形成细胞。我们首先克隆了VII型胶原基因全长9kb的cDNA，构建了该基因的表达载体，并用裸DNA方法将其导入大鼠角膜上皮细胞。结果表明，转基因后24 h在角质形成细胞中有表达，1周后在基底膜区有表达。该胶原蛋白的成功表达为DEB的基因治疗提供了可能性。此外，我们构建了IL-6表达载体，并将其导入大鼠角质形成细胞。IL-6基因导入后，肉眼可见红斑，显微镜下可见表皮肥大和淋巴细胞浸润。接下来，我们可以证明，在角质形成细胞中预先引入IL-6拮抗剂基因后，可以通过引入IL-6基因来抑制炎症。由于银屑病患者角质形成细胞中IL-6的产生增加，我们的结果提示银屑病基因治疗的可能性。在银屑病的角质形成细胞中。进一步将IL-10基因导入角质形成细胞，检测血清IL-10水平。血清中IL-10水平显著升高，循环中的IL-10可抑制皮肤远处的接触性超敏反应。这一结果提示了利用角质形成细胞作为生物反应器进行系统性疾病的基因治疗。此外，我们开发了新的角质形成细胞基因转移方法，并在DEB患者中发现了几种突变。