Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa
Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa
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DOI：
10.1038/sj.jid.5700117
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发表时间：
2006-04-01
影响因子：
6.5
通讯作者：
Shimizu, Hiroshi
中科院分区：
文献类型：
--
作者：
Goto, Maki;Sawamura, Daisuke;Shimizu, Hiroshi
营养不良性大疱性表皮松解症 (DEB) 是一种遗传性水疱性皮肤病，由 VII 型胶原蛋白基因 (COL7A1) 突变引起。将 COL7A1 引入皮肤细胞为治疗 DEB 带来了重大希望。本研究的目的是建立一种将 COL7A1 有效逆转录病毒转移到 DEB 表皮角质形成细胞和真皮成纤维细胞中的方法，并确定哪些基因转移细胞能够最有效地在皮肤中表达 VII 型胶原蛋白。我们证明，使用水疱性口炎病毒的 G 蛋白假型逆转录病毒载体和逆连蛋白的组合进行基因转移，将 COL7A1 引入缺乏 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式反应分析表明，表皮中COL7A1的表达量显着高于真皮。随后，我们用基因转移或未经处理的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，一系列的皮肤移植实验表明，与基因转移的角质形成细胞相比，基因转移的成纤维细胞向新的真皮-表皮连接处提供了更多量的VII胶原蛋白。超微结构研究表明，来自基因转移细胞的 VII 型胶原蛋白形成了适当的锚定原纤维。这些结果表明，与角质形成细胞相比，成纤维细胞可能是 DEB 治疗更好的基因治疗靶点。
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.