人紫杉醇耐药细胞株LoVo/TAX的建立及其耐药机制研究

目的:以体外建立的人结肠癌紫杉醇（paclitaxel，TAX）耐药细胞株LoVo/TAX为模型，探讨结肠癌紫杉醇获得性耐药潜在的分子机制。方法:对LoVo亲本细胞及LoVo/TAX耐药细胞进行对比分析，采用光镜及透射电镜观察细胞形态，MTT法检测细胞药物敏感性，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方法和Western blot方法分别检测多药耐药相关基因、微管蛋白相关基因以及凋亡和细胞周期调控相关基因的转录和翻译水平。结果:与亲本细胞相比，LoVo/TAX对紫杉醇高度耐药，细胞形态也有明显变化，其G2/M期阻滞降低(P<0.05)，凋亡率显著下降(P<0.01)，MRP、MAP-Tau和CDK1基因表达水平提高(P<0.05)。结论:多药耐药相关蛋白MRP1、微管相关蛋白MAP-Tau以及细胞周期蛋白依赖性激酶1（CDK1）过表达可能是引起结肠癌细胞对紫杉醇耐药的主要原因。

Establishment of paclitaxel-resistant colon cancer cell line LoVo/TAX and its drug-resistance mechanisms

Objective:To explore the potential molecular mechanism of paclitaxel resistance in colon cancer with Taxol-resistant colon cancer LoVo/TAX cell line compare with its parental cell LoVo.Methods:The morphological features were observed by inverted microscope and transmission electron microscope，drug resistance of LoVo and LoVo/TAX was determined using an MTT assay，cell cycle analysis and apoptosis were determined by flow cytometry，the mRNA levels of Mdr1，Mrp1，GST-π，LRP，TopoⅡ，TUBB3，Cdk1，MAP-Tau，Bcl-2，TP53 were assessed by real time quantitative PCR，the protein expression level of MRP1，Bcl-2，TUBB3，Cdk1 and MAP-Tau were detected by Western blot.Results:The resistance of LoVo/TAX cells to Taxol was 42.32-fold than that of the original LoVo cells.It also exhibited high-level cross-resistance to Adriamycin (ADM) and 5-fluorouracil (5-FU).The proportion of cells in G2/M phase decreased significantly (P<0.05) and the apoptosis rate of LoVo/TAX cells was also decreased significantly (P<0.01) than that of LoVo cells after treated with 1.5 μmol/L Taxol for 24 h.Overexpression of MRP1，MAP-Tau and CDK1 was associated with the development of drug resistance in LoVo/TAX.Conclusion:Overexpression of the multidrug resistance-associated protein MRP1，microtubule-associated protein (MAP)-Tau and cyclin-dependent kinase 1 (CDK1) might contribute to acquired paclitaxel resistance in colon cancer cell.