多西他赛诱导肺腺癌A549/DTX细胞株的建立及耐药机制分析

目的 建立人肺腺癌细胞A549多西他赛（DTX）耐药细胞株，并对其耐药机制进行初步分析。方法 采用逐步增加多西他赛浓度、间歇诱导的方法，建立人肺癌A549/DTX体外耐药细胞模型；MTT法检测A549/DTX的耐药特性；流式细胞仪比较多西他赛对耐药细胞株A549/DTX及亲本细胞株A549凋亡的影响；Western blot分析多西他赛作用后两种细胞凋亡调节因子Bc1-2、Bax表达差异。结果 MTT显示A549/DTX的多西他赛耐药指数为18.5；流式细胞分析显示经12.5、25、50 μg/L多西他赛作用24 h后，A549/DTX的细胞凋亡率为(6.2±4.1)％、(13.6±2.7)％、(20.5±5.1)％，A549细胞凋亡率为(15.7±3.5)％、(28.5±2.9)％和(33.1±4.8)％，两者比较差异有统计学意义（P＜0.01）；Western blot显示，与A549相比，A549/DTX细胞Bcl-2蛋白表达明显升高，Bax蛋白明显降低(P＜0.01)。结论 逐步增加浓度、间歇诱导的方法建立了稳定、耐药性较高的A549/DTX细胞株，并认为凋亡受抑是A549/DTX的耐药机制之一，与Bcl-2表达上调、Bax表达下调相关。

Establishment of Docetaxel Resistant Variant of Human Lung Adenocarcinoma Cell Line A549/DTX and Its Resistance Mechanism

Objective To establish a human lung adenocarcinoma cell line A549 with the characterizationof docetaxel (DTX) resistance, and investigate its biological mechanism of drug resistance. Methods ADTX resistant human lung adenocarcinoma cell line A549/DTX was obtained discontinuously by graduallyincreasing doses of DTX. The drug resistance of A549/DTX was evaluated by MTT assay. Apoptosis ratesof DTX resistant human lung adenocarcinoma cell line A549/DTX and parental cell line A549 were detectedby fl ow cytometry (FCM). The expression levels of Bc1-2 and Bax protein of the two cel1 1ines inducedby different concentrations of DTX were detected by Western blot. Results Resistance index(IR) ofA549/DTX was 18.5. The apoptosis rates induced by 12.5, 25 and 50 μg/L of DTX were (6.2±4.1)％, (13.6±2.7)％ and (20.5±5.1)％ in A549/DTX cells, signifi cantly lower than that in A549 cells which were (15.7±3.5)％, (28.5±2.9)％ and (33.1±4.8)％ respectively（P＜0.01）. Western blot showed a statistically higherexpression of Bc1-2 and lower expression of Bax in A549/DTX than that in A549 cells at protein levels (P＜0.01) .Conclusion A drug-resistant cell line A549/DTX with high IR was established by discontinuouslyinduction and gradually increasing doses of DTX. Apoptosis inhibition is one of the mechanisms underlyingthe drug resistance of human lung adenocarcinoma cell line A549/DTX and is related with the up-regulationof Bc1-2 expression and down-regulation of Bax expression.