人鼻咽癌耐药细胞株的建立及其与LRP的关系

Establishment of Human Nasopharyngeal Carcinoma Drug-resistant Cell Line TWO3/DDP and he Relationship of Drug Resistance with the Lung Resistance Protein

目的:建立人鼻咽癌多药耐药细胞株TW03/DDP并研究其与肺耐药相关蛋白(LRP)的关系。方法:以顺铂(DDP)为诱导药物,诱导人鼻咽癌细胞TW03,建立鼻咽癌多药耐药细胞株TW03/DDP;MTT法检测药物敏感性,流式细胞术检测细胞周期的改变,并绘制细胞生长曲线、测定倍增时间,观察细胞形态学变化;通过免疫细胞化学法、免疫印迹法、RT-PCR检测LRP的表达结果:TW03/DDP对顺铂、5-氟尿嘧啶、阿霉素、阿糖胞苷、长春新碱等药物的耐药指数分别是40.85、5.65、26.68、1.60与28.19,其半数抑制浓度与TW03比较差异有统计学意义(P<0.05);耐药细胞株的细胞倍增时间相对于其亲本细胞略有延长,其G_0/G_1期明显减少而G_2/M期则相对增加;免疫细胞化学、免疫印迹法、RT-PCR检测显示LRP及其mRNA在TW03/DDP中的表达明显高于TW03,差异具有统计学意义(P<0.05)。结论:本研究建立了人鼻咽癌多药耐药细胞株TW03/DDP,其LRP在蛋白水平及imRNA水平的表达高于亲本细胞,表明LRP的表达上调与其耐药机制有密切关系。

Objective： To investigate the relationship between drug resistance and the lung resistance protein （ LRP ） by establishing a human nasopharyngeal carcinoma drug-resistant cell line. Methods： Cisplatin resistance was induced in human nasopharyngeal carcinoma cell line TW03 by continuous exposure to gradually increasing doses of cisplatin. The resistance index of the resulting cell line, TW03/DDP, was estimated by the MTT assay. The cell cycle was examined by flow cytometry. Cell growth, doubling time, and cellular appearance were also investigated. LRP expression was measured by immunocytochemisty, Western blot, and reverse transcriptase polymerase chain reaction. Results： The resistance indices of TW03/DDP to cisplatin, 5-fluorouracil, adriamycin, cytrarabine, and vincristine were 40.85, 5.65, 26.68, 1.60, and 28.19, respectively. The proliferation of TW03/DDP was slower than TW03. The dou- bling time lengthened. The cell number of the GjM-phase increased, whereas that of the G2/M-phase decreased, compared with TW03. LRP mRNA and LRP were both up-regulated in TW03/DDP compared with TW03. Conclusion： Cisplatin-resistant cell line TW03/ DDP was established, and exhibited a typical phenotype resistant to anti-cancer drugs. LRP expression in TW03/DDP was higher than in TW03. The drug resistance of TW03/DDP correlated with the up-regulation of LRE .