华蟾素诱导U937细胞凋亡及其作用机制

Cinobufacini induces the apoptosis of U937 cells and its mechanism

目的：研究华蟾素诱导白血病U937细胞凋亡，探讨其可能的作用机制。方法：MTT法检测细胞存活率，瑞氏染色及荧光染色观察细胞凋亡形态学改变，琼脂糖凝胶电泳观察DNA片段化改变，TdT原位标记计算凋亡率，流式细胞术检测bcl-2表达，半定量RT-PCR检测Fas和Fas-1 mRNA水平。结果：与对照组相比，0．225～1．8μg/mL华蟾素作用1-3d明显抑制U937细胞生长，具有剂量．时间相关性，24h时的IC50为1．36μg/mL。0．9μg/mL华蟾素作用1d，U937细胞出现凋亡典型形态学改变；DNA电泳出现凋亡特有“梯子”条带。0．225、0．45和0．9μg/mL华蟾素作用1d，TdT原位标记检测凋亡率分别为4．8％、13．57％和24．33％。凋亡细胞bcl-2表达及Fas-1 mRNA水平下降，Fas mRNA水平增高。结论：华蟾素可能通过抑制bcl-2、Fas-1基因及活化Fas基因的途径来抑制U937细胞生长并诱导凋亡。

Objective：To investigate the effect of cinobufacini on apoptosis of U937 cells and its possible mechanism. Methods：Cell viability was measured by MTT assay. The morphological changes were observed by Wright＇s staining and immunofluorescence staining. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptotic rate was evaluated by teminal deoxynucleotidyl transferase （TdT） labeling in situ. Expression of bcl-2 protein was analyzed by flow cytometry. The levels of Fas and Fas-1 mRNA were measured by semi-quantitative reverse transcriptase polymerase chain reaction （RT-PCR）. Results：Compared with the control group, treatment with cinobufacini at 0. 225 to 1.8 μg/mL for 1-3 d significantly inhibited growth of U937 ceils in a time and dose dependent manner. The IC50 value was 1.36 μg/mL at 24 h. The typical morphological changes of apoptosis and typical apoptotic DNA ladder were observed after incubation with cinobufacini at 0. 9 μg/mL for 1 d. The apoptotic rate evaluated by TdT labeling in situ were 4. 8%, 13.57%, and 24. 33% after exposure to cinobufacini at 0. 225,0.45, and 0. 9 μg/mL for 1 d, respectively. The expression levels of bcl-2 protein and Fas-1 mRNA significantly decreased and the expression of Fas mRNA significantly increased in apoptotic cells. Conclusions：Cinobufacini inhibits growth and inducs apoptosis of U937 cells via inhibition ofbcl-2 and Fas-1 genes and activation of Fas gene.