球体形成实验联合药物筛选建立耐顺铂胰腺癌干细胞株

目的 探索建立顺铂耐药人胰腺癌干细胞株,并进行初步鉴定.方法 通过球体形成实验联合顺铂药物筛选建立耐顺铂胰腺癌干细胞株;MTT法计算耐药指数;免疫荧光半定量检测OCT-4、SOX-2表达水平,检测干细胞标记物;免疫荧光半定量检测MUC-1表达水平,检测细胞分化能力;通过流式细胞仪测量侧群细胞和表面特异抗原标记（CD44＋/CD24＋）检测肿瘤干细胞含量.结果 耐顺铂干细胞株的耐药指数是亲代株PANC-1的71.5倍;干细胞标记物的免疫荧光检测发现,耐药株中OCT-4、SOX-2、MUC-1均有不同程度的表达;耐药干细胞株及亲代株SP细胞比例分别为（25.1±1.1）％和（7.8士0.9）％,差异有统计学意义（t=6.89,P＜ 0.01）;耐药株及亲代株中CD44＋/CD24＋表型的细胞亚群分别占（20.8±1.3）％和（2.4±0.8）％,差异有统计学意义（t=16.13,P＜ 0.01）.结论 球体形成实验联合顺铂药物筛选能建立耐顺铂胰腺癌细胞株,该细胞株能高效富集胰腺癌肿瘤干细胞.

Establishment of cisplatin-resistant pancreatic cancer stem cells through sphere-formation as- say and drug screening

Objective To establish cisplatin-resistant pancreatic cancer stem cells from PANC-1 and iden- tify the cancer stem cell preliminarily. Methods Cisplatin-resistant pancreatic cancer stem cells were obtained through sphere-formation assay and drug screening in culture medium for 4 weeks. Resistance index of the cells was measured by MTT. Protein expressions of OCT-4 and SOX-2 were determined by immunofluorescence. Cellular differentiative capacity was detected based on the expression of MUC-1. Side population analysis and CD44±/CD24~ cells subsets exploration were performed by flow cytometry to examine cancer stem cells proportion. Results Cisplatin-resistant cancer cells were obtained which were 71.5 times increased in drug resistance relative to parental cells. To varying degree, resistant cell had expressions of OCT-4, SOX-2 and MUC- 1. Flow cytometry analysis showed that SP cell proportion in resistant cell was （25.1 ± 1.1）%, whereas in parental PANC-1 was （7.8 ± 0.9）%, the difference was statistically significant （t=6.89, P〈0.01）; CD44＋/CD24＋ cells was （20.8 ± 1.3）% in resistant cell, whereas （2.4 ± 0.8）% in PANC-1, the was significant difference between them （t=16.13, P〈0.01）. Conclusion To combine sphere-formation assay with drug screening can establish drugresistance pancreatic cancer stern cell line. Ciaplatin-resistant cell line has a higher proportion of pancreatic cancer stem cells compared with its parental cell line PANC-1.