Novel Tissue Models of Junctional Epidermolysis Bullosa to Characterize Functional Mechanisms of Sulfur Mustard Injury to Human Skin

摘要

We first measured dose-time responses in 2-D and 3-D cultures (TASK 1) and discovered that an SM dose of 150 micronmeter induced significant apoptotic cell death. We next compared the SM response of 3-D cultures grown in the absence or presence (AlloDerm) of structured basement membrane (BM) (TASK 8) and found that the presence of BM led to resistance to SM-induced damage, suggesting that BM could protect basal keratinocytes from SM-induced apoptosis. To further explore the role of BM in decreased SM susceptibility, primary keratinocytes harvested from Junctional Epidermolysis Bullosa (JEB) patients (no. 552), that lack a functional gamma(exp 2) chain of laminin 5 and do not adhere to BM, were transduced with retroviral vectors (TASK 4) to restore or abrogate laminin 5-mediated adhesion. We constructed 3-D tissues with these 'reverted' JEB cells (TASK 5,6 and 7) and their phenotypic analysis showed that only JEB cells with restored laminin 5 function (F-GAL) were resistant to apoptosis when exposed to SM (150 micronmeter), thereby implicating laminin 5- mediated attachment as being important in limiting SM damage. These studies provide important evidence that bioengineered, in vitro tissues mimic many skin alterations previously found in vivo and that adhesion to BM enables epithelial resistance to SM damage.