目的探讨川楝素提取物对人白血病K562细胞的增殖抑制和诱导凋亡作用及其机制。方法采用MTT法检测川楝素提取物对K562细胞增殖的影响;Wright′s染色观察细胞的形态学改变;流式细胞技术检测细胞周期与凋亡率的变化;Annexin V/PI双标记法检测细胞的早期凋亡变化;DNA琼脂糖凝胶电泳观察DNA片段化;比色法检测Caspase-3、Caspase-8、Caspase-9相对活性的改变;RT-PCR检测凋亡相关基因p21、bcr/abl、H-rasmRNA表达水平的改变。结果川楝素提取物显著抑制K562细胞的增殖,并呈剂量-时间依赖性,作用72h的IC50值为20nmol/L;处理组细胞可见典型的凋亡形态学改变;细胞周期和Annexin V/PI双标记检测表明川楝素提取物可诱导K562细胞凋亡,并呈剂量-时间依赖性;处理组细胞DNA琼脂糖凝胶电泳出现明显的DNAladder;处理组细胞Caspase-3、Caspase-8、Caspase-9的活性均显著增加;p21、H-ras mRNA表达上调,bcr/abl mRNA表达下调。结论川楝素提取物对K562细胞具有增殖抑制和诱导凋亡的作用,其机制与Caspase信号途径的活化有关。

Objective To study the effects of toosendanin extracts on the apoptosis of human erythroleukemia K562 cells.Methods MTT Assay was used to examine the effects of toosendanin extracts on the proliferation of K562 cells.Cell morphological changes were detected by Wright's stain and light microscope.Alteration of cell cycle and apoptosis rate was defected by flow cytometry. DNA Agarose gel electrophoresis was used to observe the cell apoptosis.Chromatometry was used to detect the relative activity of caspases. Expressions of p21，H-ras，and bcr/abl were analyzed by RT PCR.Results Toosendanin extracts significantly inhibited the growth of K562 cells in a dos}time dependent manner, the ICSO of 72h was 20 nmol/L.The K562 cells treated with toosendanin extracts showed morphological characteristics of apoptotic cells.Cell cycle and Annexin V/PI double labeling test indicated that toosendanin extracts-induced apoptosis of K562 cells in a concentration and time-dependent manner.The typical DNA ladder on agarose gel electrophoresis for analysis of cellular apoptosis was significantly appeared in the treated K562 cells.Toosendanin extracts induced K562 cell apoptosis involved in the activation of caspase.The mRNA expressions of p21 and H-ras were up-regulated，while the mRNA exspression of bcr/abl was down-regulated.Conclusion Toosendanin extracts could inhibit the growth and induce the poptosis of human leukemia K562 cells via caspase pathway.

重庆市科委自然科学基金计划资助项目(CSTC,2009BB5258)