筛选MDA-MB-231乳腺癌耐药细胞株两种方法的比较

Comparison of Two Methods in Establishing MDA-MB-231 Breast Cancer Resistant Cell Lines

目的 比较间歇刺激法和浓度梯度递增法对雌激素受体（estrogen receptor,ER）阴性的乳腺癌耐药细胞株的建立,为进一步探讨ER阴性乳腺癌可能的耐药机制.方法 采用间歇刺激法和浓度梯度递增法,用紫杉醇诱导6个月,分别建立了耐药细胞系231 TIM10和231 TAX8,并用倒置相差显微镜、MTT法、Western印迹、流式细胞术比较了两株耐药细胞的形态学变化、耐药表型、ER-α蛋白表达及紫杉醇诱导细胞G2/M期阻滞效应的改变等生物学特性.结果 间歇刺激法比浓度梯度递增法更容易诱导ER阴性的乳腺癌细胞获得耐药表型,231 TIM10耐药指数为11.9,231 TAX8耐药指数为2.3.用紫杉醇处理后,231 TIM10细胞能够抵抗100 nmol/L紫杉醇引起的G2/M期阻滞效应,231 TAX8细胞仅能够抵抗10 nmol/L紫杉醇引起的G2/M期阻滞效应.231 TIM10细胞在诱导过程中形成了两个细胞亚群,体积增大明显,细胞之间黏连更加明显.MDA-MB-231敏感细胞和两株耐药细胞均无ER-α蛋白的表达.结论 间歇刺激法更合理地模拟了临床上ER阴性乳腺癌的耐药过程,建立了可靠的细胞耐药模型,为耐药机制研究建立了基础.

Objective To compare two methods of pulse selection and stepwise exposure in establishment of ER-negative breast cancer resistant cell lines, and further study the resistant mechanisms of ER-negative breast cancer. Methods Paclitaxel-resistant cell lines 231 TIM10 and 231 TAX8 were established by pulse selection and stepwise exposure for 6 months. Morphological changes of the resistant cells were observed by confocal microscopy. Resistant phenotype was examined by MTF assay. Paclitaxel-induced mitotic arrest was determined by flow cytometric analysis. ER-α expression was detected by western blotting. Results 231 TIM10 variants acquired higher drug resist- ance than 231 TAX8 variants. 231 TIM10 had 11.9-fold resistance （RI = 11.9） and 231 TAX8 got 2.3-fold resistance （RI = 2.3）, compared to parental MDA-MB-231 cells. 231 TIM10 could attenuate 100nM paclitaxel-induced mitotic arrest, while 231 TAX8 could only attenuate 10 nmol/L paclitaxel-induced mitotic arrest. 231 TIM10 formed two subgroups of cell populations, showing apparently bigger size and becoming more adhesive. No ER-α expression was detected in these two resist- ant cell lines and their parental MDA-MB-231 cells. Conclusion Pulse selection reflected the clinical settings of ER-negative breast cancer acquiring drug resistance, which established reliable resist- ant cell models for further study of drug resistant mechanism of breast cancer.