Biochemical and antibacterial analysis of human wound and blister fluid
Biochemical and antibacterial analysis of human wound and blister fluid
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DOI：
10.1111/j.1432-1033.1996.0086n.x
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发表时间：
1996-04-01
期刊：
影响因子：
--
通讯作者：
Boman, HG
中科院分区：
文献类型：
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作者：
Frohm, M;Gunne, H;Boman, HG
从术后伤口、六个腿部溃疡和一个大水泡中收集液体，并通过生物化学、微生物和免疫学技术进行分析。并与血清进行比较。将所有样品冻干并用含有1%三氟乙酸的60%乙腈水溶液萃取两次。将合并的上清液冻干，再溶解，并通过六种技术（仅使用三种水泡渗出物）对液体提取物进行表征：反相HPLC，Edman降解，质谱，Western印迹分析，在具有巨大芽孢杆菌（抗Bm活性）的平板上进行抑制区测定，以及在具有来自藤黄微球菌的细胞壁的平板上进行区域清除（溶菌酶测定）。对应于伤口流体提取物的HPLC峰的材料被鉴定为：组蛋白H2 B片段1-11、1-15和1-16、完整胸腺素β-4、防御素HNP 1、2和3、溶菌酶和肽抗生素FALL-39及其前体。HPLC分离的水疱液中含有丰富的抗Bm活性物质（主要是防御素）和溶菌酶。它还可能包含以前未确定的因素。平板测定法在抗Bm活性上有50倍的差异，在溶菌酶上有10倍以上的差异，这些因子与胸腺素一起可以在伤口愈合中起作用。它的结论是，分析伤口液产生肽和活性模式与新的片段的重要肽，和定量的差异，可以是有用的，以了解伤口愈合的分子机制进一步。
Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1% trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1-11, 1-15 and 1-16, intact thymosin beta-4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated blister fluid was extremely rich in anti-Bm activity (mainly defensins) and lysozyme. It may also contain factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.