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目的:建立喉癌细胞耐药模型Hep-2/5-Fu并初步筛选可能的耐药相关基因。方法:采用5-Fu大剂量浓度逐渐递增间歇给药的方法建立人喉癌耐药细胞Hep-2/5-Fu,观察其生长规律并绘制细胞生长曲线:用MTT法鉴定其耐药性并计算耐药指数(RI);用基因芯片技术检测耐药细胞株Hep-2/5-Fu与其亲本细胞株Hep-2中的差异表达基因,从中筛选出可能的耐药相关基因;用半定量RT-PCR法进行验证。结果:Hep-2/5-Fu细胞与Hep-2细胞相比,生长缓慢,细胞体积增大,并对顺铂及长春新碱有不同程度的交叉耐药性。通过基因芯片筛选出差异表达基因1210个,可能的耐药相关基因包括Cyclin D、IGF-BP3、CASP9、CDK4/6,其中Cyclin D在Hep-2/5-Fu中的表达是亲本细胞的6.599 7倍。结论:Hep-2/5-Fu细胞株耐药性稳定,耐药机制可能与Cyclin D等耐药相关基因有关。
OBJECTIVE: To establish Hep-2/5-Fu, a drug-resistant model of laryngeal squamous cell carcinoma, and to screen for potential drug-resistance-related genes. Methods: Human laryngeal squamous cell carcinoma cell line Hep-2/5-Fu was established by increasing the dosage of 5-Fu at a high dose. The growth of laryngeal squamous cell carcinoma cell line Hep-2/5-Fu was observed and the cell growth curve was drawn. The drug resistance index (RI) was calculated. The differentially expressed genes in drug-resistant cell line Hep-2/5-Fu and its parental Hep-2 cell line were detected by gene chip technology, and the possible drug resistance- RT-PCR method to verify. Results: Compared with Hep-2 cells, Hep-2/5-Fu cells showed slow growth, increased cell volume, and different degrees of cross-resistance to cisplatin and vincristine. 1210 differentially expressed genes were screened by gene chip. The possible resistance related genes include Cyclin D, IGF-BP3, CASP9 and CDK4 / 6. The expression of Cyclin D in Hep-2/5-Fu is 6.599 7 times. Conclusion: The drug resistance of Hep-2/5-Fu cell line is stable and the mechanism of drug resistance may be related to the resistance-related genes such as Cyclin D.