Igoucheva Olga, Kelly Aislinn, Uitto Jouni, Alexeev Vitali
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Invest Dermatol. 2008 Jun;128(6):1476-86. doi: 10.1038/sj.jid.5701197. Epub 2007 Dec 13.
Junctional epidermolysis bullosa (JEB) is an inherited mechanobullous disease characterized by reduced adherence of the epidermal keratinocytes to the underlying dermis, and is often caused by the absence of functional laminin 332 due to the lack or dysfunction of its beta3 chain. As there are no specific therapies for JEB, we tested whether a protein replacement strategy could be applicable for the restoration of the laminin 332 assembly and reversion of the JEB phenotype in human keratinocytes that lack beta3 subunit. Here, we developed the protocol for production and purification of the biologically active recombinant beta3 chain. Next, we demonstrated that delivery of recombinant beta3 polypeptide into the endoplasmic reticulum of the immortalized beta3-null keratinocytes led to the restoration of the laminin 332 assembly, secretion, and deposition into the basement membrane zone, as confirmed by Western blot analysis, confocal immunofluorescent microscopy in vitro, and on cultured organotypic human JEB skin reconstructs. Although the amount of laminin 332 produced by protein-treated beta3-null keratinocytes is lower than that in normal human keratinocytes, our results demonstrate the applicability of the recombinant proteins for JEB treatment and open new perspectives for the development of novel therapeutics for this inherited, currently intractable, skin disorder.
交界性大疱性表皮松解症（JEB）是一种遗传性机械性大疱病，其特征是表皮角质形成细胞与下方真皮的黏附性降低，通常是由于β3链缺乏或功能障碍导致功能性层粘连蛋白332缺失所致。由于尚无针对JEB的特异性疗法，我们测试了蛋白质替代策略是否可用于在缺乏β3亚基的人角质形成细胞中恢复层粘连蛋白332的组装并逆转JEB表型。在此，我们开发了生产和纯化具有生物活性的重组β3链的方案。接下来，我们证明将重组β3多肽递送至永生化的β3缺失角质形成细胞的内质网可导致层粘连蛋白332的组装、分泌和沉积到基底膜带的恢复，这通过蛋白质印迹分析、体外共聚焦免疫荧光显微镜以及在培养的人JEB皮肤器官型重建物上得到证实。尽管经蛋白质处理的β3缺失角质形成细胞产生的层粘连蛋白332的量低于正常人角质形成细胞中的量，但我们的结果证明了重组蛋白在JEB治疗中的适用性，并为开发针对这种遗传性、目前难以治疗的皮肤病的新型疗法开辟了新的前景。
