人结直肠癌耐奥沙利铂细胞株的建立及特性

Establishment and characteristics of oxaliplatin-resistant human colorectal cancer cell lines

目的建立人结直肠癌耐奥沙利铂细胞株,检测该细胞株的多药耐药性及其可能的机制。方法在人结直肠癌细胞株SW480(亲本细胞)的培养液中,逐渐提高化疗药物奥沙利铂(Oxa)浓度,建立稳定的耐药细胞株(SW480/OxR),采用细胞增殖试剂WST-1检测细胞对化疗药物Oxa和5-FU的敏感性;实时荧光定量PCR检测MDR-1、Survivin及Oct4的mRNA表达水平;流式间接法检测P-gp、Survivin蛋白阳性细胞比例;Western blot方法检测Oct4蛋白表达。结果 WST-1检测细胞增殖结果为:耐药细胞加药(Oxa及5-FU)72h后细胞数量变化不大(P>0.05);耐药细胞MDR-1、Survivin和Oct4的mRNA水平,P-gp+及Survivin+细胞比例以及Oct4蛋白水平均明显高于亲本细胞(P<0.01)。结论耐药细胞具有多药耐药特性,可能与高表达耐药蛋白P-gp、抗凋亡蛋白Survivin及干细胞转录因子Oct4有关。

Objective To establish a stable oxaliplatin-resistant human colorectal cell lines and investigate the multi-drug resistance of these cell lines and the possible mechanisms.Methods Oxaliplatin-resistant（SW480/OxR） cells were generated by culturing SW480 cell lines（named as parental cells） with the increased oxaliplatin（Oxa） concentrations.The sensitivity of cells to Oxa and 5-FU was evaluated using the WST-1 reagent.The mRNA expressions of MDR-1,Survivin and Oct4 were analyzed by real-time quantitative PCR（RT-qPCR）.The percentage of P-gp（drug-resistance associated protein）-and Survivin（anti-apoptotic protein）-positive cells was measures by indirect method of flow cytometry.The Oct4（stem cell related transcription factor） protein level was detected by Western blotting assay.Results Seventy-two h after exposure to Oxa and 5-FU,the number of SW480/OxR cells was not apparently changed（P 〉 0.05）,while the number of the parental cells was significantly reduced（P 〈 0.01）.The mRNA expressions of MDR-1 and Survivin,the percentage of P-gp-and Survivin-positive cells and the mRNA and protein levels of Oct4 in SW480/OxR cells were significantly increased than those in the parental cells（P 〈 0.01）.Conclusion SW480/OxR cells exhibit characteristics of multi-drug resistance,which might be associated with the high expressions of P-gp,Survivin and Oct4.