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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IC50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G0/G1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 5-fluorouracil (5-FU) -resistant human laryngeal carcinoma cell line and to provide a model for studying the mechanism of drug resistance in laryngeal carcinoma. Methods: The multidrug resistance Hep-2/5-FU cell line Hep-2 was induced by intermittent dosing at a high dose, and the morphology and doubling time of the two groups were compared. MTT assay IC50 and drug resistance index. Flow cytometry was used to detect the cell cycle distribution and intracellular accumulation of rhodamine. The expression of MDR1 mRNA was detected by real-time quantitative PCR. The expression of MDR1-P protein was detected by Western blotting. Results: Hep-2/5-FU resistant cell lines were established with different degrees of cross-resistance with cisplatin and vincristine, and the doubling time of Hep-2/5-FU cells was higher than that of Hep- Cell prolongation [(31.25 ± 4.37) h vs (25.62 ± 3.53) h, P <0.05]. The proportion of G0 / G1 phase in Hep-2/5-FU cells was significantly higher than that in control group [(45.6 ± 3.4)% vs (30.5 ± 1.2)%, P <0.05] 52.4 ± 3.6)%, P <0.05]. Rhodamine in Hep-2 cells was significantly higher than that in Hep-2/5-FU cells [(89.83 ± 0.52)% vs (14.38 ± 0.48)%, P <0.01] (13.69 ± 1.12) vs (17.82 ± 0.61), P <0.05]. The MDR1-P protein level in Hep-2/5-FU cells was higher than that in Hep-2 cells. Conclusion: The Hep-2/5-FU cell line has a clear and stable multi-drug resistance and provides a model for the study of drug resistance mechanism of laryngeal carcinoma.