人乳腺癌多西他赛耐药细胞株BT549/Docetaxel干细胞分选及三磷酸腺苷结合转运蛋白G超家族成员2基因表达水平

摘要

目的 分选人乳腺癌多西他赛(Docetaxel)耐药细胞株BT549/Docetaxel干细胞,探讨其生物学特征.方法 采用悬浮培养联合多西他赛法分选耐药细胞株BT549/Docetaxel干细胞,采用悬浮培养法富集BT549、BT549/Docetaxel干细胞,进行CD44、CD24细胞标记和流式细胞仪检测,反转录-聚合酶链反应(RT-PCR)法检测BT549、BT549/Docetaxel干细胞中三磷酸腺苷结合转运蛋白G超家族成员2 (ABCG2)基因表达.结果 BT549/Docetaxel与BT549干细胞的群体倍增时间分别为(39.22 ±0.65)、(29.20 ±0.50)h,差异有统计学意义(P＜0.05).流式分析显示,BT549微球体细胞的CD44 +/CD24-/low比例为(13.8±0.5)％,高于单层培养BT549细胞的比例(2.8±0.6)％(P＜0.01).BT549/Docetaxel微球体细胞的CD44 +/CD24-/low比例为(19.1±0.3)％,高于单层培养BT549/Docetaxel细胞的比例(5.0±0.4)％(P＜0.01).BT549/Docetaxel微球体细胞的CD44+/CD24-/low比例高于BT549微球体细胞的CD44 +/CD24-/low比例,差异有统计学意义(P＜0.05).BT549干细胞内药物的含量为(46.9 ±0.5)ng/106个细胞,BT549/Docetaxel干细胞内药物的含量为(7.1±0.3)ng/106个细胞,两者比较差异有统计学意义(P＜0.01).正常培养基培养4h后,BT549细胞内药物的含量为(28.3±0.6) ng/106个细胞,BT549/Docetaxel细胞内药物的含量(3.5±0.1) ng/106个细胞,两者比较差异有统计学意义(P＜0.01).ABCG2的mRNA在BT549/Docetaxel干细胞内表达量明显高于在BT549干细胞内中的表达,两者差异有统计学意义(P＜0.01).结论 成功筛选人乳腺癌多西他赛耐药细胞株BT549/Docetaxel干细胞,生长及耐药性稳定.ABCG2基因在BT549/Docetaxel干细胞内表达量明显增高.%Objective To isolate docetaxel-induced human breast carcinoma drug-resistant stem cells MCF7/Docetaxel and study its biological characteristics.Methods By suspension culture combined with docetaxel resistant cell line sorting method,BT549/Docetaxel stem cells were isolated,and BT549,and BT549/Docetaxel stem cells were enriched by suspension culture method.The cells were labeled with CD44 and CD24,and detected by flow cytometry.Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of adenosine triphosphate binding cassette transporter G2 (ABCG2) in BT549 and BT549/Docetaxel stem cells.Results The population doubling time of BT549/Docetaxel and BT549 stem cells was (39.22 ± 0.65) and (29.20 ± 0.50) h with the difference being statistically significant (P＜ 0.05).Flow cytometric analysis showed CD44+/CD24-/low ratio in BT549 microspheres cells was (13.8 ± 0.5) ％,higher than that of BT549 cells in monolayer culture [(2.8 ± 0.6)％,P＜ 0.01].CD44+/CD24-/low ratio in BT549/Docetaxel microspheres cells was (19.1 ±0.3)％,higher than that in the monolayer culture [(5.0 ± 0.4)％,P＜ 0.01].CD44+/ CD24-/low ratio in BT549/Docetaxel microspheres cells was significantly higher than that in BT549 microspheres cells (P ＜ 0.05).The drug content in BT549 stem cells was (46.9 t 0.5) ng/106 cells,and that in BT549/Docetaxel stem cells was (7.1 ±0.3) ng/106 cells (P ＜0.01).After culture with normal medium for 4 h,the drug content in BT549 cells was (28.3 ±0.6) ng/106 cells,and that in BT549/Docetaxel cells was (3.5 ±0.1) ng/106 cells (P ＜0.01).The expression ABCG2 mRNA in BT549/Docetaxel stem cells was significantly higher than that in BT549 stem cells (P ＜ 0.01).Conclusion The BT549/Docetaxel stem cells were successfully isolated with stable growth and drug resistance.ABCG2 gene expression in BT549/Docetaxel stem cells was significantly increased.