Gould L J, Yager D R, McGeehan G M, Diegelmann R F
Department of Surgery, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond 23298-0117, USA.
In Vitro Cell Dev Biol Anim. 1999 Feb;35(2):75-9. doi: 10.1007/s11626-999-0004-x.
The net amount of collagen produced and deposited by fibroblasts in cell culture is determined by the rate of collagen synthesis as well as the rate of collagen degradation. Although collagen synthesis can be analyzed by several techniques, it is more difficult to measure collagen degradation. Breakdown of collagen depends upon the activity of a family of structurally and catalytically related mammalian enzymes termed matrix metalloproteinases (MMPs). Interstitial collagenase (MMP1) initiates the cleavage of fibrillar collagen, whereas gelatinases (MMP2 and MMP9) digest the denatured collagen fragments. A method has been developed to quantitate the activity of collagenase (MMP1) and gelatinase (MMP9) in conditioned medium from fibroblast cell cultures. The assay, which uses the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-AlaLys(Nma)NH2, is technically simple and amenable to high throughput analysis. Addition of specific inhibitors of the metalloproteinases allows for simultaneous measurement of both collagenase and gelatinase activity.
成纤维细胞在细胞培养中产生并沉积的胶原蛋白净量，由胶原蛋白合成速率以及胶原蛋白降解速率决定。虽然胶原蛋白合成可用多种技术进行分析，但测量胶原蛋白降解则较为困难。胶原蛋白的分解取决于一类在结构和催化方面相关的哺乳动物酶家族——基质金属蛋白酶（MMPs）的活性。间质胶原酶（MMP1）启动纤维状胶原蛋白的裂解，而明胶酶（MMP2和MMP9）消化变性的胶原蛋白片段。现已开发出一种方法，用于定量来自成纤维细胞培养条件培养基中胶原酶（MMP1）和明胶酶（MMP9）的活性。该检测方法使用荧光底物Dnp-Pro-Cha-Gly-Cys(Me)-His-AlaLys(Nma)NH2，技术上简单且适用于高通量分析。添加金属蛋白酶的特异性抑制剂可同时测量胶原酶和明胶酶的活性。
