K562/Vp16细胞系中边缘细胞对伊马替尼耐药机制的初步研究

Study on the mechanisms of imatinib-resistance of cancer stem-like cells in K562/Vp16 cell line

目的进一步阐明白血病细胞对伊马替尼耐药的机制。方法经过对K562细胞系长期的鬼臼乙叉甙(Vp16)诱导和克隆筛选,建立了一株耐药细胞系K562/Vp16,利用干细胞高效能将Hoechst33342荧光染料泵出细胞的特性,采用流式细胞术从K562/Vp16细胞系中分选出一小群细胞,即边缘细胞(Side population,SP),称为K562/Vp16SP细胞,并初步探讨了其对伊马替尼耐药的机制。结果BCR/ABL和ABL蛋白在K562细胞、K562/Vp16SP细胞及K562/Vp16非SP细胞(K562/Vp16non-SP)中的表达水平差异无统计学意义;P-gp在K562细胞中不表达,在K562/Vp16SP及K562/Vp16non-SP细胞中均高表达且表达水平一致;与K562/Vp16non-SP细胞比较,K562/Vp16SP细胞对伊马替尼的耐药性更强,并且这种耐药性几乎不能被多种多药耐药逆转剂逆转;另外,体内外实验显示,K562/Vp16细胞的致瘤性几乎全部来源于K562/Vp16SP细胞。结论白血病细胞对伊马替尼具有一定的耐药性,可能与数量极少的SP细胞有直接的关系。因此,这类数量极少的SP细胞应当成为有效治疗肿瘤的靶细胞。

Objective To elucidate the mechanisms of imatinib resistance involved in some chronic myeloid leukemia （CML） cells overexpressing P-glycoprotein （P-gp）. Methods Generation of resistant K562 cell line K562/Vp16 overexpressing P-gp was achieved by exposure of K562 cells to stepwise increase of concentrations of Vp16. A small set of side population （SP） with the characteristics of stem cells being capable of efflux fluorescent dye Hoechst 33342 in the cell line was isolated by flow cytometry. The mechanisms involved in K562/Vp16 SP cells resistant to imatinib were studied. Results The levels of BCR/ABL and ABL proteins in K562 cells were similar to those in K562/Vp16 non-SP and K562/Vp16 SP cells. The 170 KDa P-gp was detected in K562/Vp16 and K562/Vp16 SP cells at similar levels but not in K562 cells. Compared with K562/Vp16 non-SP cells, K562/Vp16 SP cells were more resistant to imatinib, which could hardly be reversed by many muhidrug resistance inhibitors. In addition, in vivo study showed that the malignancy of K562/Vp16 cells was largely attributed to the SP cells. Conclusions Bcr/Abl gene amplification and muhidrug-resistant gene 1 （ mdrl ） overexpression might not be an important clinical mechanism in the diversity of resistance to imatinib treatment, and the development of drug resistance by leukemia cells may be at least partly due to a rare SP of tumor stem-like cells which drives leukemia occurrence and maintenance. These SP cells might be targeted for effective cancer therapy.