人鼻咽癌吉西他滨耐药细胞系的建立及其生物学特性

Establishment of a gemcitabine-resistant human nasopharyngeal carcinoma cell line and its biological characteristics

目的建立人鼻咽癌吉西他滨耐药细胞系并研究其生物学特性。方法采用药物大剂量冲击法和浓度梯度递增法建立人鼻咽癌吉西他滨多药耐药细胞亚系CNE2/Gem,MTT法检测CNE2和CNE2/Gem对几种常用抗肿瘤药物的半数抑制浓度(IC50)和耐药系数(RI),流式细胞术测定细胞周期及细胞内荧光药物罗丹明的蓄积,绘制细胞生长曲线,计算倍增时间并在光镜下观察细胞形态。结果从生长曲线计算出CNE2和CNE2/Gem的倍增时间分别为17.13和23.13h。CNE2和CNE2/Gem对吉西他滨的IC50分别为2.84±1.63和42.95±7.53μg/ml,RI为23.61(P<0.001),对顺铂(DDP)、5-氟尿嘧啶(5-FU)及长春新碱(VCR)的RI分别为15.00(P<0.001)、6.98(P<0.01)及12.80(P<0.05),表明其具有多药耐药特征。流式细胞测定显示CNE2/Gem内罗丹明的蓄积明显低于CNE2(3.56vs220.35)。光镜下见CNE2/Gem胞体变圆,大小不一,胞质内有较多颗粒。结论成功建立了稳定的人鼻咽癌吉西他滨耐药细胞系CNE2/Gem,且耐药性能显著,可作为进一步研究鼻咽癌吉西他滨耐药机制较为理想的模型。

Objective To establish a gemcitabine-resistant hurrah nasopharyngeal carcinoma （NPC） cell line and study its characteristics. Methods The drug-resistant cell line CNE2/Gem was established by a procedure of treating the human NPC cells CNE2 with gemcitabine by pulse drug selection and cominuous stepwise selection. The IC50 and resistance index （RI） to several commonly used anti-tumor drugs were tested by MTT assay. Fluorescence activated cell analysis （FACS） was employed for determining the cell cycle and concentration of fluorescence dye rhodamine 123 within the cells. Cell growth curve, doubling time and cell morphology were measured and ob- served. Results Duplication time of CNE2 and CNE2/Gem was 17. 13h and 23. 13h, respectively, as evaluated by the growth curve. The IC50 to gemcitabine increased from 2. 84±1.63μg/ml in CNE2 to 42. 95±7. 53μg/ml in CNE2/Gem as tested by MTT assay at 48～72h exposure, the RI was 23. 61 （P〈0. 001）. The RI to cisplatin （DDP）, 5-fluoroutacil （5-FU） and vincristine （VCR） were 15 （P〈0. 001）, 6. 98 （P〈0. 01） and 12. 8 （P〈0. 05）, respectively, indicating its multipledrug-resistant property. FACS analysis showed that the concentration of rhodamine 123 was much lower in CNE2/DDP cells than in CNE2 cells （3. 56 vs. 220. 35）. The CNE2/Gem cells appeared roundish in various size under light microscopy, with more granulation in cytoplasm. Conclusion CNE2/Gem, the gemcitabine resistant phenotype, which showed prominem drug resistant, was established successfully. It is a good model for exploring the mechanism of gem citabine- resistant NPC.