Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers.
Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers.
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DOI：
10.1016/0003-2697(89)90711-2
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发表时间：
1989-10
影响因子：
2.9
通讯作者：
G. Boswell;S. Dimitrijevich;R. W. Gracy
中科院分区：
文献类型：
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作者：
G. Boswell;S. Dimitrijevich;R. W. Gracy
组织和伤口修复的关键步骤是去除焦痂-变性细胞和细胞外大分子的积累。使用纤溶酶（纤溶酶）和DNA酶的组合的酶促清创术已成功地用于各种类型的伤口。通过测量纤维蛋白溶解的速率或通过由于DNA水解而引起的粘度变化来监测这些酶的活性是非常麻烦、耗时的，并且充其量只是半定量的。虽然使用合成底物的分光光度测定提供了几个优点，但它们不允许将数据外推到体内遇到的更复杂的天然底物。因此，我们开发了一种体外放射性同位素测定法，用于同时定量测量纤溶酶和DNA酶的水解活性。利用在微载体珠上生长的双标记（[3 H]胸苷，[14 C]亮氨酸）人真皮成纤维细胞作为核酸和蛋白质底物的来源。该方法符合分析有效性的所有标准，灵敏、快速，适用于其他水解酶的分析。该方法提供了一个直接的评价酶清创的伤口使用实际的人类细胞基质。此外，微载体为细胞附着和生长提供了大大增加的表面积，易于通过简单的机械方法与细胞快速分离，并且理想地适合于分析操作。
A critical step in tissue and wound repair is the removal of eschar—accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered in vivo. We have, therefore, developed an in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ([3H]thymidine, [14C]leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.