目的：建立人喉癌5-氟尿嘧啶（5-fluorouracil，5-FU）耐药细胞系，为喉癌的耐药机制研究提供模型。方法：以高剂量浓度递增间歇给药的方法诱导筛选人喉癌Hep-2的多药耐药细胞株Hep-2/5-FU，比较两组细胞的形态和倍增时间；MTT法确定细胞的IC50及其耐药指数；流式细胞仪检测两组细胞的细胞周期分布以及细胞内的罗丹明聚集；实时定量PCR法检测MDR1 mRNA的表达，Western blotting检测相应MDR1-P蛋白的表达。结果：建成性能稳定的Hep-2/5-FU耐药细胞株，并与顺铂及长春新碱有不同程度的交叉耐药性，且Hep-2/5-FU细胞倍增时间较Hep-2细胞延长\[（31.25±4.37）h vs（25.62±353）h，P<0.05\]。Hep-2/5-FU细胞G0/G1期比例升高\[（45.6±3.4）% vs（30.5±1.2）%，P<0.05\]，而S期比例明显降低\[（32.1±4.2）% vs（52.4±3.6）%，P<0.05\]；Hep-2细胞内的罗丹明较Hep-2/5-FU细胞明显升高\[(89.83±0.52)％ vs (14.38±0.48)%，P<0.01\]；Hep-2/5-FU细胞MDR1 mRNA \[(13.69±1.12) vs (17.82±0.61)，P<0.05\]及其编码的MDR1-P蛋白水平高于Hep-2细胞。结论：Hep-2/5-FU细胞株具有明确及稳定的多药耐药性，为喉癌的耐药机制提供了研究的模型。

Objective:To establish a multidrug resistance cell line from human laryngeal cancer cells by 5-fluorouracil (5-Fu), and provide a research model of drug resistance mechanisms of laryngeal cancer. Methods: A multidrug resistance cell line from human laryngeal Hep-2 cancer cells (Hep-2/5-FU) was established by a high dosage intermittent increase gradient method. The morphology and cell doubling time of Hep-2 and Hep-2/5-FU cells was compared; the IC50 and drug resistance fold was detected by MTT assay; the cell cycle distribution and rhodamine accumulation in Hep-2 and Hep-2/5-Fu cells was studied by flow cytometry; and the MDR1 mRNA and the corresponding MDR1-P protein expressions were detected by real-time quantitative RT-PCR or Western blotting.Results: Hep-2/5-Fu cells was established，which had a stable resistance to 5-Fu; Hep-2/5-Fu cells exhibited cross resistance to chemotherapeutic agents like cisplatin and vincristine, and its doubling time was prolonged as compared with Hep-2 cells (\[31.25±4.37\] h vs \[25.62±353\] h, P<0.05). The proportion of cells in G0/G1 phase was increased (\[45.6±3.4\]% vs \[30.5±1.2\]%, P<0.05), while decreased in S phase (\[32.1±4.2\]% vs \[52.4±3.6\]%, P<0.05) in Hep-2/5-Fu cells as compared with those in Hep-2 cells; rhodamine accumulation in Hep-2 cells was much higher than that in Hep-2/5-Fu cells(\[89.83±0.52\]% vs \[14.38±0.48\]%, P<0.01); and the expressions of MDR1 mRNA (\[13.69±1.12\] vs \[1782±061\], P<0.05) and the corresponding MDR1-P protein were increased in Hep-2/5-Fu cells than those in Hep-2 cells. Conclusion: The Hep-2/5-Fu cell line shows the typical and stable multidrug resistance, and may serve as a research model of laryngeal cancer resistance mechanism.

上海仁济医院科研培育基金项目资助（No. RJPY10-011）