人舌鳞状细胞癌顺铂耐药细胞株CAL-27/DDP的建立及其生物学评价  

Establishment of human tongue squamous cell carcinoma cisplatin resistant cell line CAL-27/DDP and its biological evaluation

作　　者：姜慧[1,2] 张桐菲[1] 杨鹤[1] 徐召南 毕也 孙抒[2] 张泽兵[1] 贾杰[3] 

机构地区：[1]吉林大学口腔医院病理科,吉林长春130021 [2]延边大学基础医学院病理学与法医学教研部,吉林延吉133002 [3]吉林大学中日联谊医院电诊科,吉林长春130033

出　　处：《吉林大学学报（医学版）》2016年第3期506-511,I0003,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅自然基金资助课题(20150101174JC);吉林省卫计委科研基金资助课题(20132004)

摘　　要：目的:建立人舌鳞状细胞癌(舌鳞癌)顺铂耐药细胞株CAL-27/DDP,探讨其顺铂耐药机制。方法:采用顺铂浓度梯度法对人舌鳞癌CAL-27细胞株进行诱导,将细胞分为CAL-27组和CAL-27/DDP组。CAL-27组细胞株用普通培养液培养,CAL-27/DDP组细胞株用含有不同浓度顺铂的培养液培养,经过12个月的诱导形成生长良好的耐药细胞系株CAL-27/DDP。采用倒置显微镜观察细胞株形态表现;MTT法检测细胞株的半数抑制浓度(IC50)和耐药指数(RI);流式细胞术检测细胞株的细胞周期;RT-PCR法检测细胞株中多药耐药基因(MDR1)mRNA的相对表达水平;Western blotting法检测细胞株P糖蛋白(P-gp)的相对表达水平。结果:CAL-27/DDP组细胞株在一段时间内能在普通培养液中稳定生长,并维持其形态和耐药性状。与CAL-27组细胞株比较,CAL-27/DDP细胞略增大,细胞中出现颗粒和"空泡"样变,胞浆丰富,倍增时间延长(P<0.01),RI为18.09±0.30。与CAL-27组比较,CAL-27/DDP组G1期和G2期细胞比例减少,S期细胞比例升高,CAL-27/DDP组细胞株中MDR1mRNA和P-gp相对表达水平升高(P<0.01)。结论:本研究建立了人舌鳞癌顺铂耐药细胞株CAL-27/DDP,其耐药机制与CAL-27/DDP细胞株中MDR1和P-gp的表达水平上调有关联。Objective：To establish the drug-resistant cell line CAL-27/DDP of human tongue squamous cell carcinoma,and to discuss the mechanism of cisplatin resistance.Methods：The CAL-27/DDP cells were induced by increasing the concentrations of cisplatin in vitro.Then the cells were divided into CAL-27 group and CAL-27/DDP group.The cells in CAL-27 group were teated with common nutrient solution and the cells in CAL-27/DDP group were treated with nutrient solution containing different concentrations of cisplatin.The resistance cell line CAL-27/DDP with good growth status was formed 12 months after induction.The morphology of cells was observed by inverted microscope;the half maximal inhibitory concentration（IC50）value and drug resistance index（RI）of cisplatin in the CAL-27 and CAL-27/DDP cells were detected by MTT assay;the cell cycle distribution of CAL-27 and CAL-27/DDP was detected by flow cytometry;the relative expression level of MDR1 in the cells was detected by reverse transcriptase polymerase chain reaction（RT-PCR） method;the relative expression level of P-glycoprotein（P-gp）in the cells was detected by Western blotting method.Results：The cells in CAL-27/DDP group could stably grow in the ordinary medium in a period and maintained its morphology and resistance status.Compared with CAL-27 group,the cells in CAL-27/DDP group showed particles and ＂cavity＂samples,plenty of plasma,and increased doubling time（P〈0.01）;the RI was 18.09±0.30.Compared with CAL-27 group,the ratios of cells at G1 phase and G2 phase in CAL-27/DDP group were decreased,and the ratio of cells at S phase and the relative expression level of MDR1 mRNA in the CAL-27/DDP cells were increased significantly（P〈0.01）,and the relative expression level of P-gp was increased（P〈0.01）.Conclusion：The drug-resistant cell line CAL-27/DDP of human tongue squamous cell carcinoma is established sucessfully and the drug-resistant mechanism may be related to the up-regulation of MDR1 and P-gp expression levels in CAL-27/DDP

关 键 词：癌 鳞状细胞 舌肿瘤 顺铂 耐药性 MDR1 P糖蛋白 

分 类 号：R739.86[医药卫生—肿瘤]