拓扑异构酶I抑制剂诱导白血病HL60/VCR耐药细胞凋亡的研究

目的　研究拓扑异构酶I抑制剂 (topotecan ;TPT)对白血病耐药细胞HL6 0 /VCR诱导凋亡的作用。方法　瑞氏 吉姆萨染色和吖啶橙 /溴化乙啶 (AO/EB)染色进行形态观察 ,采用TUNNEL、流式细胞仪检测细胞周期和AnnexinV观察TPT对HL6 0 /VCR细胞的抑制效应和促凋亡作用。WesternBlot检测bcl 2、活化的caspase 3和P糖蛋白 (Pgp)的表达变化。 结果　经TPT处理后的HL6 0 /VCR细胞 ,在光镜和AO/EB染色中均可见到典型的凋亡细胞形态学改变 ,并具有时间和剂量依赖性 ;AnnexinV染色后能检测到早期凋亡细胞 (2 6 .8% ) ,细胞周期显示 :G1期细胞比例增高 ,S期减低 ,并有 2 1.8%凋亡峰 ;TUNNEL能检测到 (6 2 .2± 3.5 ) %的阳性细胞 ;伴有活化的caspase 3的表达和bcl 2的下调 ,而Pgp的表达在细胞凋亡前后无变化。结论　TPT能诱导白血病耐药细胞凋亡 ,该过程伴有caspase 3的活化和bcl 2的表达下调。

Apoptosis of HL60/VCR Cells Induced by Topotecan

Objective To gain insight into the mechanisms of topotecan(TPT) inducing apoptosis of acute myelogenous leukemia multidrug-resistant HL60/VCR cells. Methods Exposed HL60/VCR cells to different concentrations of TPT, morphologic evidence for apoptosis was determined by Wright-Gimsa and Acridine Orange/ethidium bromide(AO/EB) staining. Cell cycle, Sub-G1 and Annexin V FITC staining were detected by flow cytometry. The expression of active caspase-3, bcl-2 and Pgp were detected by Western blot. Results HL60/VCR cells treated by TPT are observed to have apoptosis characteristic morphological changes by Wright-Gimsa and AO/EB staining. The percentage of annexin V-positive cells was 26.8%. The apoptosis cells increased significantly with TUNNEL.The proportion of G 0/G 1 HL60/VCR cells treated by TPT was increased and the sub-G1 was 21.8%. Meanwhile, HL60/VCR cells treated by TPT expressed activated caspase-3, and the expression of bcl-2 decreased. However, the expression of Pgp didn't change. Conclusion TPT could induce apoptosis of HL60/VCR cells with appearance of active-caspase3 and lower expression of bcl-2.