氨甲蝶呤对映体耐药A549细胞的表皮生长因子受体基因表达及迁移能力

目的研究氨甲蝶呤(MTX)对映体耐药人非小细胞肺癌A549细胞的迁移能力以及表皮生长因子受体(EGFR)mRNA的表达。方法用细胞划痕试验检测L-(+)-MTX/A549细胞和D-(-)-MTX/A549细胞的迁移能力;双层软琼脂克隆试验检测L-(+)-MTX/A549细胞和D-(-)-MTX/A549细胞的克隆形成率并观察集落的形态;用RT-PCR检测亲本A549细胞、L-(+)-MTX/A549细胞和D-(-)-MTX/A549细胞中EGFR mRNA的表达。结果加入MTX 72 h后D-(-)-MTX/A549细胞的迁移能力(1 230.1±40.2)高于L-(+)-MTX/A549细胞(530.3±25.4);D-(-)-MTX/A549细胞、L-(+)-MTX/A549细胞和亲本A549细胞的克隆形成率(%)分别为(1.38±0.17)、(1.36±0.13)和(1.37±0.15),差异无统计学意义(P>0.05);亲本A549细胞、L-(+)-MTX/A549细胞均有EGFR mRNA表达,其光密度值(IDV)分别为(6 630±64)、(3 697±27),差异有统计学意义(t=103.42,P<0.01)。而D-(-)-MTX/A549细胞不表达EGFR。结论 D-(-)-MTX诱导的A549细胞的迁移能力大于L-(+)-MTX。EGFR基因表达具有手性差异。

Expression of epidermal growth factor receptor gene in methotrexate enantiomers-resistant A549 cells and influence on cellular migration ability

Objective To investigate the migration ability of methotrexate(MTX) enantiomers-resistant non-small cell lung cancer(NSCLC) cell line A549 and the expression of epidermal growth factor receptor(EGFR) in the cells.Methods The migration ability of L-(+)-MTX/A549 and D-(-)-MTX/A549 cells were evaluated by cell scratch assay.The colony formation rates and the morphology of cell cluster of L-(+)-MTX/A549 and D-(-)-MTX/A549 were determined by double-layer soft agar colony formation assay.The mRNA expression of EGFR in parental A549 cells,L-(+)-MTX/A549 cells,D-(-)-MTX/A549 cells were detected by RT-PCR.Results The migration ability of D-(-)-MTX/A549 cells(1 230.1±40.2) was stronger than that of L-(+)-MTX/A549 cells(530.3±25.4) at 72 h after adding MTT.The rate of colony formation in D-(-)-MTX/A549,L-(+)-MTX/A549 and parental A549 cells was(1.38±0.17),(1.36±0.13) and(1.37±0.15) respectively.There was no statistically significant difference among D-(-)-MTX/A549,L-(+)-MTX/A549 and parent A549 cells(P>0.05).The mRNA expression of EGFR in parental A549 cells was(6 630±64),and in L-(+)-MTX/A549 cells was(3 697±27),thus statistical difference between the two groups was found(t=103.42,P<0.01),but the EGFR mRNA in D-(-)-MTX/A549 cells barely expressed.Conclusion A549 cells induced by D-(-)-MTX showed greater migration ability than those induced by L-(+)-MTX.The expression of EGFR gene presented significant difference in different chirality.