河北医药
河北醫藥
하북의약
HEBEI MEDICAL JOURNAL
2014年
21期
3217-3219
,共3页
郝秀菊%刘志辉%栾文姬%王锋%苏丽娜%陈俊峰
郝秀菊%劉誌輝%欒文姬%王鋒%囌麗娜%陳俊峰
학수국%류지휘%란문희%왕봉%소려나%진준봉
L-鸟氨酸脱羧酶%L-鸟氨酸脱羧酶抑制剂%筛选模型
L-鳥氨痠脫羧酶%L-鳥氨痠脫羧酶抑製劑%篩選模型
L-조안산탈최매%L-조안산탈최매억제제%사선모형
L-ornithine decarboxylase%L-ornithine decarboxylase inhibitor%screening model
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。
目的:基于酶促反應法,利用HPLC測定腐胺含量,評價待篩選藥物對PC-3M細胞中L-鳥氨痠脫羧酶( L-ODC)活性的影響,建立鳥氨痠脫羧酶抑製劑的篩選模型。方法將與藥物反應後的細胞處理樣本,以TSKge lODS-80TM凝膠色譜柱(150 mm ×4.6 mm,5μm)為分析柱,在甲醇-水(8∶2)保持3 min,從4 min到14 min,甲醇比率由80%梯度增至100%梯度洗脫,流速為0.8 ml/min,激髮波長為340 nm,髮射波長為515 nm,柱溫為50℃,進樣量10μl。按內標法定量。結果 ODC特異性抑製劑二氟甲基鳥氨痠(DFMO)對ODC活性的抑製率為(59.3±5.4)%,雷帕黴素對ODC活性的抑製率為(29.8±6.4)%,薑黃素對ODC活性的抑製率為(26.1±6.6)%。結論本模型通過測定藥物對ODC細胞內鳥氨痠脫羧酶的作用,利用HPLC分離法測定在藥物抑製影響下的鳥氨痠脫羧酶的活性,這一模型有望成為篩選ODC抑製劑的快速方便準確的檢測方法。
목적:기우매촉반응법,이용HPLC측정부알함량,평개대사선약물대PC-3M세포중L-조안산탈최매( L-ODC)활성적영향,건립조안산탈최매억제제적사선모형。방법장여약물반응후적세포처리양본,이TSKge lODS-80TM응효색보주(150 mm ×4.6 mm,5μm)위분석주,재갑순-수(8∶2)보지3 min,종4 min도14 min,갑순비솔유80%제도증지100%제도세탈,류속위0.8 ml/min,격발파장위340 nm,발사파장위515 nm,주온위50℃,진양량10μl。안내표법정량。결과 ODC특이성억제제이불갑기조안산(DFMO)대ODC활성적억제솔위(59.3±5.4)%,뢰파매소대ODC활성적억제솔위(29.8±6.4)%,강황소대ODC활성적억제솔위(26.1±6.6)%。결론본모형통과측정약물대ODC세포내조안산탈최매적작용,이용HPLC분리법측정재약물억제영향하적조안산탈최매적활성,저일모형유망성위사선ODC억제제적쾌속방편준학적검측방법。
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.