喉癌顺铂耐药细胞株的建立及干性生物学特性分析

Establishment of a cisplatinresistant human laryngeal carcinoma cell line and its stem cell biological characteristics

摘要:

摘要：目的建立人喉癌顺铂（CDDP）耐药细胞株并鉴定其干细胞生物学特性，探讨其对CDDP耐药机制及与干细胞的关系。方法以药物浓度梯度递增法诱导建立人喉癌Hep2的顺铂耐药细胞株Hep2/CDDP；倒置显微镜观察细胞的形态；CCK8法检测细胞IC50及耐药指数；无血清细胞球体培养观察成球能力；实时定量RTqPCR法检测MDRl/Pgp、ABCG2、CD44、CD133 mRNA的表达情况；Western blot法检测MDRl/Pgp、ABCG2、CD44、CD133蛋白表达情况。结果顺铂体外缓慢诱导历时12个月建成的Hep2/CDDP耐药细胞株，与亲本Hep2细胞相比，细胞形态改变明显；耐药性能稳定耐药指数为5.43；无血清球体培养实验示细胞成球率升高(P<0.05)；RTqPCR和Western blot结果表明耐药相关基因MDRl/Pgp、ABCG2表达上调(均P<0.05)；干性相关标记基因CD44、CD133升高(均P<0.05)。结论Hep2/CDDP细胞株具有稳定的顺铂耐药性，是研究喉癌顺铂耐药和逆转机制的良好模型，且其耐药机制与顺铂诱导的肿瘤干细胞有关,可为研究逆转耐药提供参考。

关键词:顺铂|喉癌|耐药|干细胞

Abstract:

Abstract:ObjectiveTo establish a cisplatinresistant cell line from human laryngeal carcinoma and identify its stem cell biological characteristics for study on the mechanism of cisplatin resistance and its relation with the stem cells.MethodsCisplatinresistant human laryngeal carcinoma cell line Hep2/CDDP was established with induction by gradient increment method of cisplatin concentration. Cell morphology was observed with inverted microscope. The half maximal inhibitory concentration (IC50) value and drug resistant index (RI) were detected by CCK8 assay. Microsphere formation ability was detected by culturing in serumfree medium (SFM). The relative mRNA expression levels of MDRl/Pgp, ABCG2, CD44 and CD133 were detected by RTqPCR. Their protein expression levels were detected by Western blot.ResultsThe Hep2/CDDP cell line was successfully established in vitro after cisplatin induction for 12 months. Compared with parent Hep2 cells, cellular morphology of Hep2/CDDP was changed obviously. The resistant ability was stable and RI value was 5.43. Microsphere formation test indicated increased formation ratio of Hep2/CDDP (P<0.05). The expressions of resistanceassociated genes MDRl/Pgp, ABCG2 in Hep2/CDDP were upregulated significantly (all P<0.05).The expression levels of stem cellassociated gene CD44、CD133 in Hep2/CDDP were increased significantly (all P<0.05).ConclusionsThe Hep2/CDDP cell line, possessing stable cisplatin resistance, may serve as a model for study of both drugresistance and reversing mechanisms in laryngeal carcinoma. Furthermore, the mechanism of resistance is associated with cisplatininduced stem cells, which may provide reference for the study of reverse resistance.

Key words:Cisplatin|Laryngeal neoplasm| Dug resistance| Stem cell