Abstract: 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 RNaseⅠ＋DNaseⅠ, then they were seeded by myo-fibroblasts 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 serumfree 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.
Citation： HONG Hao,DONG Nianguo,SHI Jiawei.. Amplex Red Fluorometric Assay for Detection of Lysyl Oxidase in Tissue Engineered Heart Valve. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2007, 14(1): 27-30. doi: Copy