| Summary: | 碩士 === 國防醫學院 === 生物及解剖學研究所 === 98 === The idea that transplants could express immunomodulatory proteins following gene transfer to achieve immunosuppressive effect has been proposed since the last decade. Decoy receptor 3 (DcR3) belongs to tumor necrosis factor receptor (TNFR) superfamily that lacks the transmembrane domain and is found overexpressed in many kinds of tumors. DcR3 blocks both FasL- and LIGHT-induced cell deaths and modulates immune responses. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into free iron, biliverdin, and carbon monoxide. By preventing of oxidative damage and via a local immunomodulatory influence on infiltrating inflammatory cells, HO-1 represents a cell defense mechanism. In previous studies, both of DcR3 and HO-1 can ameliorate rejection and improve the survival of many transplants. We use an ideal gene transfer vector, lentivirus, which can transduce non-dividing cells, achieve long-term transgene expression and be non-immunogenic, to transduce tissue-engineered skin substitutes with DcR3 and HO-1 in order to investigate the anti-inflammatory effect. The transduced efficiency, gene expression and protein production were estimated by immunofluorscence staining, flow cytometry, and western blot. Results indicated that the lentiviral transduction rate for keratinocytes was in a dose- and time-dependent manner, and was nearly 90%. The expression and production of DcR3 was detectable in supernatants of transduced cells culture. Transduced cells were confluent on the collagen/polycaprolactone biocomposite films and kept their properties. To explore the cytoprotection against FasL, we demonstrate that expression of DcR3 and HO-1 suppresses FasL-mediated apoptosis by MTT assay and flow cytometry. The immunomodulatory effects of transduced cells on cytokine production of immune cells were estimated by ELISA. The expression of IL-2 and IFN-γwere both induced in co-culture system, whereas the expression of IL-10 remained un-affected. The up-regulation of IL-10 and down-regulation of IL-2 were observed in transduced cell.
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