摘要
目的 :研究国产氨肽酶 N抑制剂 bestatin对人白血病细胞的作用及其机理。方法 :应用 MTT比色法观察 bestatin对细胞的生长抑制作用 ,流式细胞仪检测细胞表面 CD13的表达 ;光学显微镜观察细胞形态结构的改变 ;DNA凝胶电泳、DNA片段原位末端标记及流式细胞仪 (FCM)检测 ,以分析细胞凋亡 ;Rho-damin(Rh) 12 3染色后 ,FCM检测细胞线粒体跨膜电位。结果 :(1) Bestatin明显抑制 HL 6 0细胞的生长 ,半数抑制 (IC5 0 )约为 13.0 3μg/ ml,而 K5 6 2细胞的敏感性较差 ,IC5 0 大于 40 0μg/ ml,其抑制作用均呈剂量 -时间效应关系 ;(2 )典型的细胞形态改变 ,DNA片段化 ,DNA末端原位标记的检出及 FCM结果 ,均证实 bestatin能诱导白血病细胞的凋亡 ;(3) 10 μg/ m l bestatin作用 2 4h即可明显诱导 HL6 0细胞凋亡 ,K5 6 2细胞的凋亡敏感性显著低于 HL6 0细胞 ,两者凋亡率均呈剂量和时间依赖性 ;(4 )经 bestatin作用后 ,细胞出现 G1期阻滞 ;(5 )经bestatin处理的 HL 6 0细胞出现线粒体跨膜电位 (△Ψ m)下降。结论 :Bestatin能抑制 K5 6 2、HL 6 0细胞生长 ,诱导凋亡是其机制之一 ,该效应可能与线粒体△Ψ
Objective: To study the mechanism of actions of bestatin inhibiting human leukemic cells. Methods: K562 and HL60 cells were treated with bestatin at different concentrations and growth inhibition phases and were analysed by MTT assay. Cell apoptosis was evaluated by light microscopy, agarose gel electophoresis, TUNEL labeling method and flow cytometry (FCM) assay. The mitochondrial transmembrane potentials (△Ψm) were detected through staining of Rhodamine 123, of which fluorescent intensities were measured on FCM. Results: 1. Treatment with bestatin remarkably inhibited the growth of HL60 cells, the IC 50 value of bestatin for HL60 cells was found to be 13.03μg/ml. But K562 cellswere less sensitive than HL60, the IC 50 value for K562 cells was more than 400μg/ml. Bestatin inhibited the growth of HL60 and K562 cells in a dose time dependent manner; 2. Apoptpsis of leukemic cells could be induced by bestatin, showing the apoptotic changes in morphology, DNA ladder on agarose gel, labeled by TUNEL method, apoptotic peak before G1 phase of cell cycle; 3. Bestatin induced apoptosis in K562 and HL60 cells in a dose time dependent manner; 4. The cell cycle analysis measured by FCM showed that the cells were in G1 blocking after treated with bestatin; 5. The △Ψm of cells collapse was induced by bestatin. Conclusion: Bestatin could efficiently induce apoptosis in HL60 and K562 cells, and this is one of the mechanisms for inhibiting leukemia of bestatin, which could be associated closely with the △Ψm collapse induced by bestatin.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2001年第3期218-221,共4页
Chinese Journal of Antibiotics
