| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 98 === Currently, the treatment of skin burning injury is graft surgery. But the patient with a large area skin burning injury do not have enough skin for surgeay. Therefore, it’s potential for the research and developing of new skin burning injury dressings. Here we use a biocomposite membrane made by collagen type I, and combine with the primary culture keratinocyte . The biocomposite membrane can provide protection and help healing the injury. But the primary culture keratinocyte will induce immune attack and cause allograft rejection. Therefore, we use Adeno associated virus(AAV) to modify the keratinocyte for overexpressing Decoy receptor 3(DcR3) and Heme oxygenase-1(HO-1). Heme oxygenase-1 is an inducible rate-limiting enzyme that catalyzes heme into carbon monoxide(CO), free iron, and biliverdin. Recently studies show that HO-1 have the function of anti-inflammatory, anti-oxidation, and anti-apoptosis. Moreover, it is known that HO-1 activates IKK and NF-κB, the activation is depend on Fe2+-mediated ROS formation. Increased nuclear binding of NF-κB induces the expression of c-FLIPs which rescues the cells from Fas-triggered death signaling by blocking procaspase-8 cleavage.DcR3 is one of the tumor necrosis factor receptor (TNFR) family, and it belongs to the third kind of TNFR. The third kind of TNFR lack cytoplasmic domain. There major function is binding to other TNF and block the signaling pathway. DcR3 is a soluble, secreted protein, the relation between DcR3 level and tumor development is quite close. DcR3 can bind Fas ligand (FasL)to block Fas-mediated apoptosis signaling from cytotoxic T-lymphocyte. DcR3 can also bind LIGHT, and TL1A to block T-lymphocyte activation. We want to develop a new kind of biocomposite membrane that have anti-immune attack effects, so it can be used for large area skin burning injury. Our result showed that after AAV infection, primary culture keratinocyte can overexpress DcR3 and HO-1. By immunocytochemistry and flow cytometry analysis , the modified keratinocyte can express both DcR3 and HO-1. In western blot, after infection 24 hours, the overexpression can be obvious observed. The overexpression continued for at least 72 hours. We use soluble FasL to mimic the apoptosis made immune attack, after FasL treatment the apoptosis of keratinocyte can be observed. The modified keratinocyte that express HO-1 or DcR3 show the reduction of apoptosis. The anti-apoptosis effect of co-infection DcR3 and HO-1 is better. Our study shows the anti-immune effects of an AAV-mediated biocomposite membrane, that can be used as a skin burning injury dressing
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