目的 建立人三阴性乳腺癌细胞耐药模型MDA-MB-231/DOX,并考察其生物学特性。方法 以人三阴性乳腺癌细胞为亲本细胞,采用低浓度持续加量诱导法建立耐受盐酸多柔比星的细胞株MDA-MB-231/DOX。在显微镜下观察细胞形态学的变化;利用细胞毒性实验检测化疗药物对细胞的半数抑制浓度(IC50)和细胞的耐药指数(RI),并绘制细胞生长曲线;通过流式细胞术检测细胞周期变化;采用Western blot 法检测细胞中P糖蛋白(P-gp)、多药耐药相关蛋白(MRP2)、乳腺癌耐药蛋白(BCRP)、葡萄糖转运蛋白(GLUT1、GLUT3)的表达;通过检测细胞中丙酮酸、乳酸、三磷酸腺苷(ATP)浓度考察MDA-MB-231/DOX细胞在能量代谢方面的生物学特性。结果 所构建的能够耐受不同浓度盐酸多柔比星的MDA-MB-231/DOX细胞,其耐药指数在61~189范围内,对紫杉醇、顺铂和10-羟基喜树碱的耐药指数分别为1 585.12、29.67、2.01;耐药细胞MDA-MB-231/DOX中P-gp、MRP2的蛋白表达水平明显高于亲本细胞MDA-MB-231,丙酮酸浓度、乳酸浓度、ATP浓度、GLUT1表达均高于亲本细胞MDA-MB-231。结论 成功构建了人三阴性乳腺癌耐药细胞株MDA-MB-231/DOX,具有多药耐药性,其生物学特性与亲本细胞存在一定差异,为乳腺癌体外模型及其耐药机制的研究提供实验基础。
Abstract
OBJECTIVE To establish a doxorubicin hydrochloride(DOX) resistant cell line model of human triple-negative breast cancer MDA-MB-231/DOX and investigate its biological characteristics. METHODS Doxorubicin hydrochloride-resistant breast cancer cell line was established by continuous induction with a gradual increasing dose of DOX. The cell morphology changes were observed under an optical microscope; the half maximal inhibitory concentration(IC50) and RI(resistance index) of drug resistant cells to chemotherapeutics were examined by cytotoxicity experiments and the cell growth curve was plotted; the cell cycle distribution was determined by flow cytometry; the protein expression of P-glycoprotein(P-gp), multidrug resistance-associated protein(MRP2), breast cancer resistance protein(BCRP) and glucose transporter(GLUT1, GLUT3) in cells were examined by Western blot; the biological characteristics of MDA-MB-231/DOX cells in the aspect of energy metabolism were investigated by detecting the concentration of pyruvic acid, lactic acid and adenosine triphosphate(ATP) in cells. RESULTS The established MDA-MB-231/DOX resistant cell line was resistant to different concentrations of DOX, and its resistance index to DOX was in the range of 61-189. The resistance indexes of MDA-MB-231/DOX cells to paclitaxel(PTX), cisplatin(CDDP) and 10-hydroxycamptothecin(HCPT) were 1 585.12, 29.67 and 2.01, respectively; the protein expressions of P-gp and MRP2 in drug-resistant MDA-MB-231/DOX cells were significantly higher than those in parental MDA-MB-231 cells; the concentrations of pyruvic acid, lactic acid and ATP, and the protein expression of GLUT1 in MDA-MB-231/DOX cells were also higher than that in MDA-MB-231 cells. CONCLUSION The human triple-negative breast cancer resistant cell line MDA-MB-231/DOX was successfully established. The cell line exhibits multiple drug resistanc and its biological characteristics are different from the parental cells to some extent. The present research will provide some experimental basis for establishing breast cancer model in vitro and exploring the related drug resistance mechanism.
关键词
三阴性乳腺癌 /
盐酸多柔比星 /
能量代谢 /
耐药 /
MDA-MB-231/DOX细胞株
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Key words
triple negative breast cancer /
doxorubicin hydrochloride /
energy metabolism /
drug resistance /
MDA-MB-231/DOX cell line
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中图分类号：
R965.1
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脚注
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基金
国家自然科学基金资助(81873198);上海市科委优秀学术带头人项目资助(19XD1423700);上海中医药大学产业发展中心医养结合科创项目计划资助(YYKC-2021-01-160)
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