小鼠黑色素瘤细胞系B16F10中侧群细胞的分离及其耐药性初探

Sorting of the side population cells from mouse melanoma cell line B16F10 and the preliminary analysis on the mechanism of drug resistance

利用荧光活化细胞分选技术,我们分选小鼠黑色素瘤B16F10细胞系中的侧群细胞(SP细胞)和非侧群细胞(Non-SP细胞),用噻唑兰(MTT)法检测化疗药长春新碱对两群细胞的抑制率,计算半数抑制浓度(IC50),并通过RT-PCR和荧光定量PCR法比较两者ABCG2基因表达量。结果显示,B16F10细胞系中存在0.96%的SP细胞,化疗药长春新碱对SP细胞的IC50是1.26μg/ml,对Non-SP细胞的IC50是0.37μg/ml,两者差异有显著性意义(P<0.05)。荧光定量PCR法检测SP细胞的ABCG2分子表达量是Non-SP细胞的6.27倍。结果提示,小鼠黑色素瘤细胞系B16F10中存在SP细胞,其较Non-SP细胞显示出更强的耐药性,SP细胞较高表达ABCG2可能是其耐药机制之一。

To sort the side population cells（SP cells） and non-side population cells（non-SP cells） from mouse melanoma cell line B16F10 and to investigate the mechanism of drug resistance of chemotherapy drug oncovin,the SP and Non-SP cells from B16F10 cell line were sorted by fluorescein activated cell sorter（FACS）.The 50% concentration of inhibition（IC50） to oncovin was determined by using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide and the expression of ABCG2 molecule was analyzed by RT-PCR and real-time-PCR assay.It was found that the SP and non-SP cells could be sorted by FACS,and 0.96% of SP cells were detected in B16F10 cells line.The IC50 of the SP cells to oncovin was 1.26 μg/ml,whereas that of the non-SP cells was 0.37 μg/ml.This difference showed statistical significance（P0.01）.The expression quantity of ABCG2 molecule in SP cells was about 6.27 times more than that in non-SP cells.These results indicate that the SP cells in mouse melanoma cell line B16F10 showed more resistance to chemotherapy drug oncovin than that in non-SP cells,and the high expression of ABCG2 molecule on the SP cells may be related to the mechanism of drug resistance.