人结肠癌紫杉醇耐药细胞株的建立及耐药相关基因mRNA表达的研究

Establishment of Paclitaxel-Resistant Colon Cancer Cell Line and the m RNA Expressions of Drug Resistance-Related Genes

目的建立紫杉醇耐药细胞株LoVo/Taxol,探讨结肠癌细胞紫杉醇获得性耐药的可能机制。方法反复用紫杉醇处理结肠癌LoVo细胞,诱导建立结肠癌紫杉醇耐药细胞株LoVo/Taxol;通过MTT法检测细胞药物敏感性,光学显微镜和透射电镜观察细胞形态学变化,流式细胞术检测细胞周期分布及细胞凋亡水平,实时荧光定量PCR法测定细胞中多药耐药基因MDR1、MRP1、LRP、GST-π和TopoⅡ的表达。结果建立的紫杉醇耐药细胞株LoVo/Taxol对紫杉醇的耐药指数为42,为高度耐药;对阿霉素及5-氟脲嘧啶也产生强烈的交叉耐药,耐药指数分别为796和187。相较于亲本细胞LoVo,LoVo/Taxol细胞形态发生明显的变化,G2/M期阻滞和细胞凋亡率明显降低(P<0.05)。在五种经典的耐药相关蛋白中MRP1、MDR1基因m RNA的转录水平分别提高了37倍和2倍,LRP、GST-π基因m RNA的转录水平分别下降了11倍和3倍(P均<0.05),To Po II基因转录水平无明显变化。结论成功建立的人结肠癌紫杉醇耐药细胞株LoVo/Taxol为典型的多药耐药细胞模型,其耐药机制可能与MRP1基因的过表达有关。

Objective To establish a paclitaxel resistant cell line LoVo/Taxol and to investigate the mechanisms of Taxol resistance in colon cancer cells.MethodsDrug-resistant cell line LoVo/Taxol was induced by exposing LoVo parent cells to moderate concentration of Taxol. The IC50 and resistant index（RI）to Taxol was determined by MTT assay.The morphological changes of LoVo and LoVo/Taxol was observed by fluorescence microscope and transmission electron microscopy,cell cycle distribution and apoptosis of cell lines were examined by flow cytometry while the mRNA expressions of mdr1,mrp1,lrp1,GST-πand TopoⅡgenes was detected by qRT-PCR.ResultsA paclitaxel-resistant human colon cancer cell line,LoVo/Taxol,was successfully established. Compared with the parent cell line LoVo,it was 42-fold resistant to paclitaxel,and 696 and 187-fold resistant to Doxorubicin and 5-fluorouracil,respectively（P〈0.05）. Moreover,the proportion of G2/M phase decreased significantly（P〈0.05）,meanwhile,the apoptosis rate of LoVo/Taxol cells was also decreased significantly（P〈0.01）than that of LoVo cells after treated with 1.5μmol·L-1 Taxol for 24h. Over-expression of MRP1 was associated with the development of drug resistance in LoVo/Taxol. ConclusionA stable and paclitaxel-resistant human colon cancer cell line LoVo/Taxol was successfully established and the mechanisms of its drug resistance might be related to the overexpression of the multidrug resistance-associated protein MRP1.