吉西他滨耐药人胰腺癌细胞株的建立及其与肿瘤干细胞的相关性研究

Establish a gemcitabine-resistant pancreatic cancer cell line SW1990/GZ and research the relationship between SW1990/GZ and pancreatic cancer stem cell

目的 建立吉西他滨耐药人胰腺癌细胞株SW1990/GZ,并探讨SW1990/GZ和胰腺癌肿瘤干细胞的相关性.方法 应用间歇浓度梯度倍增法建立吉西他滨耐药人胰腺癌细胞株SW1990/GZ;倒置显微镜下观察细胞形态;MTT法计算耐药指数（RI）;荧光定量PCR检测ABCB1、ABCC1及ABCG2基因的表达水平;裸鼠皮下种植瘤试验观察SW1990和SW1990/GZ的成瘤能力;流式细胞仪通过侧群细胞（SP）法和表面特异抗原标记法（CIM4＋CD24＋）检测肿瘤干细胞含量.结果 在形态学上,SW1990/GZ较SW1990发生明显改变;SW1990/GZ的耐药指数是亲代SW1990的77.2倍;与亲代SW1990相比,耐药株SW1990/GZ中ABCB1、ABCC1及ABCG2的表达水平明显增高（P〈0.01）,裸鼠皮下成瘤能力增强（P〈0.01）;耐药株SW1990/GZ中SP细胞比例为（11.0±1.0）%,亲代SW1990中SP细胞比例为（4.6±0.9）%,CD44＋CD24＋细胞在两者中的比例分别为（8.7±0.8）%和（1.1±0.4）%（P〈0.01）.结论 吉西他滨耐药胰腺癌细胞株SW1990/GZ能高效富集胰腺癌肿瘤干细胞,CD44与胰腺癌获得性耐药关系密切,可能为克服胰腺癌获得性耐药提供新的治疗靶点.

Objectives To establish a gemcitabine-resistant pancreatic cancer cell line SW1990/ GZ,and to explore the relationship between drug-resistant cell line SW1990/GZ and pancreatic cancer stem cell. Methods Gemcitabine-resistant pancreatic cancer cell line SW1990/GZ was obtained by treating parental cell line SW1990 in vitro with increasing dosage of gemcitabine in culture medium intermittently for 24 weeks. Stable cultures were obtained which were 77. 2-fold increased in resistance relative to parental cells. Gene expressions of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP were determined by real-time PCR. Tumorigenic potential was performed by nude mice xenograft transplant experiments. Side population analysis and CD24CD44 positive cells explore were determined by flow cytometry to examine cancer stem cell proportion. Results Gemcitabine-resistant cell line SW1990/GZ underwent obvious morphological and functional changes. Compared with the parental cell line,SW1990/GZ cell was small and turned into round shape. SW1990/GZ had a higher gene expression level of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP than SW1990（P 〈0. 01）. Nude mice xenograft transplant experiments showed that only 1 x 105 SW1990/GZ cells were sufficient for tumor formation, whereas an injection of 1 x 105 SW1990 cells did not initiate tumors. Flow cytometry analysis showed that SP proportion in SW1990/GZ was （11.0±1.0）%, whereas in parental SW1990 it was （ 4. 6 ± 0. 9 ） % , CD44CD24 positive cells was （8. 73±0. 81） % in SW1990/GZ, whereas （1.1±0. 4）% in SW1990. Conclusions Gemcitabine-resistant cell line SW1990/GZ has a higher proportion of pancreatic cancer stem cells compared to its parental cell line SW1990. CD44 is mainly responsible for acquired drug resistance,which can be a potential target to overcome acquired drug resistance in pancreatic cancer.