L-鸟氨酸脱羧酶抑制剂筛选模型的建立

摘要

目的：基于酶促反应法，利用HPLC测定腐胺含量，评价待筛选药物对PC-3M细胞中L-鸟氨酸脱羧酶（ L-ODC）活性的影响，建立鸟氨酸脱羧酶抑制剂的筛选模型。方法将与药物反应后的细胞处理样本，以TSKge lODS-80TM凝胶色谱柱（150 mm ×4．6 mm，5μm）为分析柱，在甲醇-水（8∶2）保持3 min，从4 min到14 min，甲醇比率由80％梯度增至100％梯度洗脱，流速为0．8 ml/min，激发波长为340 nm，发射波长为515 nm，柱温为50℃，进样量10μl。按内标法定量。结果 ODC特异性抑制剂二氟甲基鸟氨酸（DFMO）对ODC活性的抑制率为（59．3±5．4）％，雷帕霉素对ODC活性的抑制率为（29．8±6．4）％，姜黄素对ODC活性的抑制率为（26．1±6．6）％。结论本模型通过测定药物对ODC细胞内鸟氨酸脱羧酶的作用，利用HPLC分离法测定在药物抑制影响下的鸟氨酸脱羧酶的活性，这一模型有望成为筛选ODC抑制剂的快速方便准确的检测方法。%Objective To evaluate the effect of screening drugs on activity of L-ornithine decarboxylase ( L-ODC) in PC-3M cells by means of HPLC,and to establish the screening model of L-ornithine decarboxylase inhibitor.Methods A TSKge lODS-80TM column (150mm ×4.6mm,5μm) was used for chromatographic separations,in gradient conditions at 0.8ml/min,with the mobile phase composed by water-methanol.The initial mobile phase was composed by 80% methanol, 20%water,maintained for 3min.Then during 4~14min,the composition was linear changed to 100%methanol,maintained for 3 min.The excitation wavelength was set at 340nm and emission wavelength was 515nm.The injection volume was 10μl. Results The inhibition rate of difluoromethylornithine (DMFO) to the activity of L-ODC was(59.3 ±5.4)%,the inhibition rate of rapamycin to activity of L-ODC was (29.8 ±6.4)%and the inhibition rate of curcumin to the activity of L-ODC was (26.1 ±6.6)%.Conclusion The model established by detecting the effect of L-ornithine decarboxylase in cells and detecting the activity of L-ornithine decarboxylase by means of HPLC may become a rapid, accurate, convenient detection method for screening ODC inhibitor.