Amplex Red荧光法检测组织工程瓣膜赖氨酰氧化酶活性

Amplex Red Fluorometric Assay for Detection of Lysyl Oxidase in Tissue Engineered Heart Valve

目的采用Amplex red荧光法检测组织工程心脏瓣膜(tissue engineered heart valve,TEHV)中赖氨酰氧化酶(lysyl oxidase,LOX)的活性。方法经胰蛋白酶+乙二胺四乙酸、聚乙二醇辛基苯基醚和核酸酶处理,去除猪主动脉瓣叶的细胞成分,然后在其表面种植大鼠肌成纤维细胞,构建TEHV。用高糖培养基在体外培养瓣膜27 d,再用不含酚酞和血清的培养基培养24h,收集此时的培养基作为样本,用Amplex red荧光法检测样本中LOX的浓度,同时应用逆转录-聚合酶链反应(RT-PCR)检测TEHV中LOXmRNA的表达。结果样本中LOX的荧光值为45.60±1.66,根据标准曲线获得相应的LOX浓度为0.123±0.003μg/ml,同时各个TEHV均有LOXmRNA的表达,与Amplex red荧光法检测的结果相一致。结论应用Amplex red荧光法检测TEHV中LOX的活性具有可行性,并为反映LOX对TEHV细胞外基质中胶原交联的重要作用提供了方法依据。

Objective Using Amplex red fluorometric assay to detect the lysyl oxidase （LOX） enzyme activity in tissue engineered heart valve （TEHV）. Methods Porcine aortic valves were decellularized with trypsin＋ethylene diaminetetraacetic acid （EDTA）, TritonX-100, and RNase I＋DNase 1 , then they were seeded by myofibroblasts that harvested from rats. Then they were fed with Dulbecco＇s modified Eagle medium （DMEM） which contained high glucose for 27 days, they were fed with phenol red-free and serum-free DMEM for 24 hours, and the medium was harvested and used for LOX enzyme activity assays with the Amplex red fluorometric assay. And reverse transcription-polymerase chain reaction （RT-PCR） technique was used to analyze the expression of LOXmRNA in TEHV. Results All the samples produced measurable amounts of active LOX enzyme. The fluorescence units were 45. 60＋1. 66, and the corresponding concentration of LOX enzyme were 0. 123＋0. 003μg/ml. At the same time, all the samples expressed LOXmRNA. The expression of LOXmRNA was corresponding to the results of the Amplex red fluorometric assay. Conclusion It is feasible to detect the LOX enzyme activity in TEHV with the Amplex red fluorometric assay. And this assay gives a way to reflect that LOX plays an important role in collagen cross-linking of extracellar matrix in TEHV.