Goto Maki, Sawamura Daisuke, Ito Kei, Abe Masataka, Nishie Wataru, Sakai Kaori, Shibaki Akihiko, Akiyama Masashi, Shimizu Hiroshi
Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
J Invest Dermatol. 2006 Apr;126(4):766-72. doi: 10.1038/sj.jid.5700117.
Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal-epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.
营养不良性大疱性表皮松解症（DEB）是一种由VII型胶原蛋白基因（COL7A1）突变引起的遗传性皮肤水疱病。将COL7A1导入皮肤细胞进行治疗对DEB的治疗具有重要前景。本研究的目的是建立一种将COL7A1高效逆转录病毒转染至DEB表皮角质形成细胞和真皮成纤维细胞的方法，并确定哪种基因转染细胞能在皮肤中最有效地表达VII型胶原蛋白。我们证明，使用水疱性口炎病毒G蛋白假型逆转录病毒载体和纤连蛋白的组合进行基因转染，可将COL7A1导入一名缺乏COL7A1表达的DEB患者的角质形成细胞和成纤维细胞中。对正常人皮肤的实时聚合酶链反应分析表明，表皮中COL7A1的表达量明显高于真皮。随后，我们用基因转染或未处理的DEB角质形成细胞和成纤维细胞制作了皮肤移植物，并将其移植到裸鼠体内。有趣的是，一系列皮肤移植实验表明，基因转染的成纤维细胞比基因转染的角质形成细胞向新的真皮-表皮交界处提供了更多的VII型胶原蛋白。超微结构研究显示，来自基因转染细胞的VII型胶原蛋白形成了合适的锚定原纤维。这些结果表明，成纤维细胞可能是比角质形成细胞更好的DEB治疗基因治疗靶点。
