| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 94 === Most of cobra snakebites have been shown to induce significant local tissue necrosis and with a retarded wound healing process in addition to systemic neurotoxic paralysis.We used one step SPFF chromatography to purify atragin from the venom of Naja atra. On fibroblast migration in the wound closure assay ,40nM atragin inhibited~30% wound healing at 24 h without significant cytotoxicity. We also tested the effect of CTX A5 in the same assay, 5μM CTX A5 inhibited~40% of wound closure at 24 h compared to control without detectable cytotoxicity. Interesting, 5μM CTX A5 and 40nM atragin synergistically inhibited~65% of wound closure compared with 30% inhibition of 40nM atragin alone. However,this phenomena might come from cell cytotoxicity. Apoatragin, which lacked metalloprotease activity, is unable to inhibit fibroblast migration. We checked the secondary structure and αvβ3 integrin binding activity of apoatragin that ensure structural and functional stability. CHO cells expressing recombinant αvβ3 (β3-CHO) adhere to atragin-coated surface in a dose-dependent manner at the levels higher than the control or α5-CHO cells andβ3-CHO cells cells are more susceptible to atragin compared with wildtype- andα5- variants. Finally, atragin may impair fibroblast interaction with ECM protein via affecting cell surface receptor to binding to αvβ3 integrin ligand ,(fibronectin, vitronectin and fibrinogen ), but not to collagen. Taken together, atragin inhibited fibroblast migration through digesting ECM and cell surface receptor such asαvβ3 integrin.
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