人乳腺癌细胞MCF-7紫杉醇耐药株的建立及其生物学特性研究

目的建立紫杉醇(Taxol)的人乳腺癌耐药细胞株MCF-7/Taxol,并初步研究其生物学特性。方法采用低浓度加量持续诱导法建立MCF-7/Taxol;细胞模型的多药耐药性以SRB法检测;流式细胞术分析细胞周期分布;免疫组化法检测细胞表型变化;高效液相鉴定细胞中药物蓄积;光镜和电子扫描显微镜观察细胞形态。结果SRB法检测,MCF-7/Taxol细胞的紫杉醇半数抑制浓度(IC50)是亲代MCF-7细胞的525倍,撤药3mon后细胞的耐药指数保持在150倍以上,并对羟喜树碱、表柔比星、多柔比星、米托蒽醌、硫酸长春新碱、博来霉素和丝裂霉素等多种化疗药物产生交叉耐药性。MCF-7/Taxol细胞分化程度低于同步传代的MCF-7细胞,倍增时间明显高于亲本细胞,S期细胞明显增加,G1期细胞减少;随撤药时间的延长,细胞的增殖速度加快。MCF-7/Taxol细胞P-gp、LRP和GSTπ的表达水平较亲本细胞有明显增加,ER、PR阳性表达丢失;光镜下MCF-7/Taxol细胞明显变大并且形态不规则;电镜下其表面纤绒毛成小球状隆起和絮状突起;在稳定生长的耐药细胞和撤药培养的细胞中都有Taxol蓄积。结论该MCF-7/Taxol模型具有多药耐药细胞的基本生物学特性,可用于肿瘤多药耐药机制药的研究,推测耐药细胞株中含有天然MCF-7肿瘤干细胞。

Establishment and characteristics of a paclitaxel resistant human mammary adenocarcinoma cell subline (MCF-7/Taxol)

Aim To establish a new paclitaxel resistant human mammary adenocarcinoma cell subline(MCF-7/Taxol) and investigate its characteristics.Methods A paclitaxel resistant human mammary adenocarcinoma cell subline(MCF-7/Taxol) was developed by gradually increasing the concentration of Taxol from the parent cell line MCF-7 in vitro.The multidrug resistance of MCF-7/Taxol to anticancer agents was evaluated by SRB assay;the distribution of their cell cycles was detected by flow cytometry;the positive expression rate of P-gP、LRP、ToPoII、GSTπ、ER and PR was measured by S-P immunohistochemistry;the intracellular accumulation of Taxol was assessed by HPLC;the morphological features and the celluar ultrastructure characteristics were observed respectively by light microscopy and scanning electron microscopy.Results MCF-7/Taxol was resistant to several chemotherapy agents,such as Hydroxycamptothecine,Epirubicin,Doxorubicin,Mitoxantone and so on.The IC50 of MCF-7/Taxol to Taxol was 525 times higher than that of MCF-7,and the IC50 of MCF-7/Taxol stopped administrating Taxol for three months was 150 times higher than that of MCF-7.The multiplication time of MCF-7/Taxol was longer than that of the natural cell and the proportion of cells in S-phase increased while that in G1-phase decreased.The expression levels of P-gp、LRP and GSTπ increased,and ER and PR were not observed.The morphology of MCF-7/Taxol became larger and irregular,and the surface of natural cell was in the shape of floss,but MCF-7/Taxol was in the shape of bead.The intracellular of Taxol was observed in both MCF-7/Taxol and stoppage administrating.Conclusions MCF-7/Taxol cell subline was a typical multidrug resistant cell line which had basic characteristics of drug resistance cells.It was supposed that there was a cell subline which was tumor stem cell of MCF-7 included in this multidrug resistant cell line.