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目的:探讨紫杉醇诱导筛选的喉癌Hep-2耐药细胞的多药耐药特性,为逆转喉癌的多药耐药提供理论依据。方法:以浓度逐渐递增法诱导筛选喉癌Hep-2细胞的耐药细胞株Hep-2T,比较2种细胞的形态和倍增时间。以ATP酶检测法确定细胞的IC50和细胞的耐药倍数。流式细胞仪检测2种细胞的周期分布情况、细胞凋亡以及2种细胞内的罗丹明聚集情况。以实时定量RT-PCR法检测MDR1和MRP1基因,Western-botting法检测相应的蛋白表达情况。结果:耐药细胞株的耐药倍数是非耐药株的104倍。耐药株对多柔吡星、吉西它宾、5-Fu及顺铂的耐药倍数分别是非耐药株的46.78,1.95,2.50和1.05倍。与非耐药株比,耐药细胞G0/G1期的细胞较后者升高(P<0.05),而G2/M和S期细胞则较后者明显降低(P<0.05)。当作用于Hep-2的IC50浓度时,Hep-2细胞的凋亡细胞数较Hep-2T明显高。罗丹明染色显示,Hep-2细胞内的罗丹明较Hep-2T明显增高。Hep-2T细胞MDR1/GAPDH表达在基因和蛋白水平明显高于Hep-2细胞。MRP1/GAPDH在基因和蛋白水平的表达前者亦高于后者,但增高程度不如MDR1/GAPDH明显。结论:在寻找逆转喉癌多药耐药的方法时,应着重于MDR1/P-gp,当应用化疗药时,应考虑非P-gp作用底物的药物。并选择应用周期特异性作用的药物。
Objective: To investigate the multidrug resistance of paclitaxel-induced laryngeal carcinoma Hep-2 cells and to provide a theoretical basis for reversing multidrug resistance of laryngeal carcinoma. Methods: The drug-resistant cell line Hep-2T of laryngeal carcinoma Hep-2 cells was induced by increasing the concentration gradually. The morphology and doubling time of the two kinds of cells were compared. The IC50 and cell multiples of the cells were determined by ATPase assay. The cell cycle distribution, apoptosis and aggregation of rhodamine in two kinds of cells were detected by flow cytometry. Real-time quantitative RT-PCR detection of MDR1 and MRP1 gene, Western-blotting method to detect the corresponding protein expression. Results: The resistant rate of drug-resistant cell lines was 104 times than non-resistant strains. The resistant rates of multidose, gemcibibine, 5-Fu and cisplatin were 46.78, 1.95, 2.50 and 1.05 times of those of non-resistant strains respectively. Compared with the non-resistant strains, the number of cells in G0 / G1 phase was higher than that in the latter (P <0.05), while the cells in G2 / M and S phase were significantly lower than those in the latter (P <0.05). When treated with Hep-2 at IC50 concentration, the number of apoptotic cells in Hep-2 cells was significantly higher than that in Hep-2T cells. Rhodamine staining showed that rhodamine in Hep-2 cells was significantly higher than Hep-2T. Hep-2T cells MDR1 / GAPDH expression in gene and protein levels was significantly higher than Hep-2 cells. MRP1 / GAPDH at the gene and protein levels of the former is also higher than the latter, but not as high as MDR1 / GAPDH. CONCLUSIONS: MDR1 / P-gp should be focused on reversing the multidrug resistance of laryngeal carcinoma. When using chemotherapeutic agents, drugs that are non-P-gp acting substrates should be considered. And select the application of cycle-specific drugs.