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

doi:10.13481/j.1671-587x.20160317

摘要/Abstract

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。

关键词: 癌, 鳞状细胞, 舌肿瘤, 顺铂, 耐药性, MDR1, P糖蛋白

Abstract:

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 mechanismmay be related to the up-regulation of MDR1 and P-gp expression levels in CAL-27/DDP cells.

Key words: carcinoma,squamous cell, tongue neoplasms, cisplatin, drug resistance, MDR1, P-glycoprotein

中图分类号:

R739.86

姜慧, 张桐菲, 杨鹤, 徐召南, 毕也, 孙抒, 张泽兵, 贾杰. 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[J]. 吉林大学学报(医学版), 2016, 42(03): 506-511.

JIANG Hui, ZHANG Tongfei, YANG He, XU Zhaonan, BI Ye, SUN Shu, ZHANG Zebing, JIA Jie. Establishment of human tongue squamous cell carcinoma cisplatin resistant cell line CAL-27/DDPand its biological evaluation[J]. Journal of Jilin University Medicine Edition, 2016, 42(03): 506-511.

参考文献

[1] Wamakulasuriya S.Global epidemiology of oral and oropharyngeal cancer[J].Oral Oncol,2009,45(4/5):309-316.

[2] 邱嘉旋,魏军水,朱声荣,等.Ki-67、EGFR、P53及RARβ 在口腔鳞癌化疗前后的变化及临床意义评价[J].口腔医学研究,2011,27(2):127-131.

[3] Vikulova IV.The state-of-the-art of the diagnosis of oral and oropharyngeal cancer[J]. Vestn Rentgenol Radiol,2012(3):51-54.

[4] 张建忠,柯樱,沈佳琳.铂类抗肿瘤药物的研发进展及市场情况[J].上海医药,2013,34(23):52-59.

[5] Tong X,Dong S,Yu M,et al.Role of heteromeric gap junctions inthe cytotoxicity of cisplatin[J].Toxicology, 2013,310:53-60.

[6] Mendoza J, Martinez J, Hernandez C, et al. Association between ERCC1 and XPA expression and polymorphisms and the response to cisplatin in testicular germ cell tumours[J].Br J Cancer,2013,109(1):68-75.

[7] Li X,Ling V,Li PC.Same -single -cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip[J].Anal Chem,2008,80(11):4095-4102.

[8] Sakaeda T, Kadoyama K, Okuno Y. Adverse event profiles of Platinum agents: data mining of the public version of the FDA adverse event reporting system, AERS, and reproducibility of clinical observations[J].Int J Med Sci,2011,8(6):487-491.

[9] 肖扬,王万春.姜黄素对人骨肉瘤细胞株/阿霉素多药耐药逆转作用的实验研究[J].卫生研究,2011,40(1):103-106.

[10] Goldstein L.Clinical reversal of drug resistance[J].Curr Prob Cancer,1995,19(2):18-21,70-80,83-106,109-123.

[11] Jang SH, Wientjes MG, Au JL. Kinetics of P-glycoprotein-mediated efflux of paclitaxel.[J]. J Pharmacol Exp Ther,2001,298(3):1236-1242.

[12] 延冰,刘国勤,姜永胜,等.高剂量法诱导胃癌铂类耐药细胞株的建立及其生物学特性[J].山东大学学报:医学版,2014,52(4):49-52,57.

[13] 黄在菊,杨守华,李敏,等.不同方法建立卵巢癌紫杉醇耐药细胞株对临床用药方案的启发[J].中国药师,2008,10(11):1135-1137.

[14] Findling-Kagan S,Sivan H,Ostrovsky O,et al.Establishment and characterization of new cellular lymphoma model expressing transgenic human MDR1 [J]. Leuk Res,2005,29(4):407-414.

[15] 张峰,岑娟.肿瘤多药耐药模型的建立与评价方法[J].药物评价研究, 2013,36(5):377-381.

[16] 张浩轩,孙小锦,孙一鸣,等.氯喹逆转人鼻咽癌细胞HNE1/DDP的耐药作用[J].南方医科大学学报,2015,35(5):687-691.

[17] 周丽佳,杨洲,张士博,等.载顺铂聚乳酸静电纺丝膜对口腔鳞癌细胞的杀伤作用研究[J].中国实验诊断学,2014,18(2):179-182.

[18] 徐亚娟,孙旭,李烁烯,等.CKS1 siRNA对人舌癌Tca 8113细胞中CKS1蛋白的抑制作用[J].中国老年学杂志,2015,35(3):743-745.