【摘要】 目的　探讨半边旗提取物 6F的细胞毒作用及诱导DNA片段化与蛋白激酶C(PKC)信号转导途径的关系 ,检测 6F对PKC活性的影响。方法　受试对象为HL 6 0细胞 ,超速离心法获得的胞液 (可溶部分 )及颗粒 (不可溶部分 ,包括细胞膜系统及胞核 )部分用作PKC活性测定。经 0 .4g·L- 1磷脂酰丝氨酸 ,0 .0 4g·L- 1甘油二油酸酯激动剂作用酶粗提物后 ,用液体闪烁计数仪计数 [γ 32 P]ATP参入外源底物的量以测定PKC活性。MTT法测定HL 6 0细胞的活力 ,二苯胺法测定 6F诱导DNA片段化程度。结果　在所测试的浓度范围内 (0 .5～ 312 μmol·L- 1) ,化合物 6F显著抑制胞液及颗粒部分PKC活性 ,最大抑制率达 88.6 % ,呈浓度依赖关系 (胞液部分r =0 .781,P <0 .0 5 ,颗粒部分r =0 .931,P <0 .0 1)。 6F诱导HL 6 0细胞DNA片段化及对细胞的毒性作用可被具有致癌作用的PKC激活剂肉豆蔻酸酯 (PMA ,浓度为 6 5nmol·L- 1)拮抗 ,抑制率分别是 30 %和 4 4% (P <0 .0 1)。PMA单独用使HL 6 0细胞线粒体将MTT还原为甲月赞的能力增强 14 % (P <0 .0 1) ,即增强细胞活力。结论化合物 6F是PKC的抑制剂。 6F对HL 6 0细胞DNA片段化的诱导作用及其细胞毒作用至少可部分归因于其对PKC活性的抑制作用更多还原

【Abstract】 AIM To investigate whether compound 6F isolated from Pteris semipinnata L inhibits the activity of protein kinase C(PKC) and whether the DNA fragment induction and cytotoxicity of 6F on HL 60 cells relate to PKC signaling pathway. METHODS HL 60 cells were used as in vitro model and its cytosol(soluble sample) and particle(insoluble sample including membrane system and nuclei) fractions obtained by ultracentrifugation were used as samples for PKC assay. PKC activity was measured by incorporation of [γ 32 P]ATP into exogenous substrate after stimulated by phosphatidylserine and diolein. Diphenylamine assay and MTT staining methods were applied for DNA fragmentation detection and cytotoxicity assay, respectively. RESULTS PKC activities in cytosol and particle fractions were inhibited by 6F in a dose dependent manner(P<0.05 in the series of cytosol fraction, P<0.01 in the series of particle fraction). Phorbol 12 myristate 13 acetate(PMA,65 nmol·L -1 ) partially attenuated the DNA fragmentation induced by 6F in HL 60 cells, and protected cells against the cytotoxic effect of 6F(P<0.01). The ability of reducing MTT to formazan in mitochondria of HL 60 cells was potentiated when the cells were treated with PMA alone at the same concentration for 24 h(P<0.01). CONCLUSION Compound 6F is an inhibitor for PKC. DNA fragment induction and cytotoxic effect of 6F may be partially through its inhibitory effect on PKC activity.更多还原