5-氟尿嘧啶对肝癌细胞株PLC/RAF/5生物学特性及耐药机制的影响

Effects of 5-fluorouracil on biological characteristics and drug resistance mechanisms of liver cancer cell line PLC/RAF/5

目的研究化疗药物5-氟尿嘧啶(5-Fu)反复作用肝癌细胞株PLC/RAF/5后生物学特性的变化,探讨耐药肝癌细胞与肝癌干细胞之间的相关性。方法用低浓度的5-Fu(1μg/ml)反复多次作用于人肝癌细胞株PLC/RAF/5后建立PLC/RAF/5/5-Fu细胞株,观察两种细胞的形态学差异。用CCK-8法检测不同浓度的5-Fu(0、0.25、0.5、1、1.5、2μg/ml)对两种细胞增殖的抑制作用;在不同浓度的5-Fu(0.5、1、2μg/ml)作用48 h后,流式细胞仪分析不同浓度下两种细胞的凋亡情况;检测两种细胞中侧群细胞的比例;平板克隆集落实验比较两种细胞的克隆形成能力;Western Blot检测两种细胞中Bax、Bcl-2、ABCG2和Fox M1蛋白的表达情况。计量资料组间比较采用t检验。结果用化疗药物5-Fu成功建立PLC/RAF/5/5-Fu细胞株。PLC/RAF/5/5-Fu细胞比PLC/RAF/5细胞体积大,突起减少,呈长梭形改变,折光性增强。与亲本细胞相比,PLC/RAF/5/5-Fu细胞对5-Fu增殖抑制作用的敏感性降低,细胞周期G0/G1期比例降低,而S期和G2/M期比例上升;抗凋亡能力增加;侧群细胞比例增加;增殖能力增强,上述差异均具有统计学意义(P值均<0.05)。Western Blot检测表明PLC/RAF/5/5-Fu细胞Bcl-2、Fox M1、ABCG2蛋白表达增强,而PLC/RAF/5细胞的Bax蛋白表达增强,差异具有统计学意义(P值均<0.01)。结论细胞株PLC/RAF/5/5-Fu中肝癌干细胞可能是其耐药的根源;低浓度的5-Fu(1μg/ml)可以富集肝癌干细胞样细胞。

Objective To study the changes in biological characteristics of a liver cancer cell line PLC/RAF/5 after repeated exposure to a chemotherapy drug, 5 - fluorouraci （5 - FU） , and to investigate the relationship between drug - resistant liver cancer cells amt liver cancer stem cells. Methods A low concentration of 5 - FU （ 1 μg/ml） was used to treat the human liver cancer cell line PLC/RAF/5 repeatedly to establish the PLC/RAF/5/5 - FU cell line. Morphological differences between the two types of ceils were observed. The inhibitory effects of different concentrations of 5 - FU （0, 0.25,0.5, 1, 1.5, and 2 μg/ml） on the proliferation of the two types of cells were determined using the CCK - 8 assay. Apoptosis of the two types of cells after exposure to different concentrations of 5 - FU （0.5, 1 , and 2 μg/ml） for 48 h was analyzed using flow eytometry. The proportions of side population cells in both types of cells were measured using flow eytometry. The colony -forming ability was compared between the two types of cells by the plate colony -forming assay. The expression of Bax, Bcl -2, ABCG2, and FoxMl proteins in both types of cells was examined by Western blot. Between - group comparison was performed by t test. Resuits The PLC/RAF/5/5 - FU cell line was successfully established using the chemotherapy drug 5 - FU. Compared with the PLC/RAVV5 cells, the PLC/RAF/5/5 - FU cells had a larger volume, fewer protrusions, a changed shape of a long shuttle, and enhanced refractivity. Moreover, compared with the parent cells, the PLC/RAF/5/5 -FU cells had a significantly lower sensitivity to the inhibitory effect of 5 - FU on proliferation, a significantly lower proportion of cells at the GO/G1 phase of the cell cycle, significantly higher proportions of cells at the S and G2/M phases, significantly higher resistance to apoptosis, a significantly higher proportion of side population cells, and significantly enhanced proliferation （P 〈0.05）. According to the results of Western blot assay, the expression of Bel -2, FoxM1, and ABCG2 proteins in the PLC/RAF/5/5 - FU cells was significantly increased （P 〈0.01 ） , while the expression of Bax protein in the PLC/RAF/5 cells was significantly increased （P 〈 0.01 ）. Conclusion Liver cancer stem cells are probably responsible for drug resistance of the PLC/RAF/5 - FU cell line. Exposure to a low concentration of 5 - FU （ 1 μg/ml） can enrich liver cancer stem cell - like cells.