氨甲蝶呤对映体获得性耐药A549细胞株二氢叶酸还原酶基因表达分析  

Analysis for different expression of dihydrofolate reductase gene in methotrexate enantiomers-resistant lung cancer A549 cell lines

作　　者：李道静[1] 何晓东[1] 孙余婕[1] 凡任芝[1] 许维东[1] 孙利[1] 张永娟[1] 张白银[1] 沈佐君[1] 

机构地区：[1]安徽医科大学附属省立医院临床检验中心,合肥230001

出　　处：《临床检验杂志》2011年第3期219-221,共3页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金资助项目(30672011);安徽省自然科学基金资助项目(050430902);安徽省人才开发资金资助项目(2005Z040)

摘　　要：目的研究氨甲蝶呤(MTX)对映体[L-(+)-MTX和D-(-)-MTX]耐药与二氢叶酸还原酶(DHFR)基因表达的关系。方法用浓度递增结合低剂量持续诱导法获得A549细胞对不同构型及不同浓度的MTX对映体的耐药细胞株,荧光定量PCR检测耐药细胞株中DHFR基因的相对含量。结果对两种不同对映体的获得性耐药存在差异,D型耐药细胞耐药指数高于L型;对映体各浓度耐药细胞间耐药指数也有差异。15μmol/L L型、D型MTX首次诱导耐药细胞的DHFR相对含量低于亲本细胞,对该浓度对映体耐药的各细胞组间没有差别(P>0.05)。35～45μmol/L浓度耐药细胞的DHFR相对含量增加,45μmol/L D型耐药细胞DHFR相对含量高于对应浓度L型耐药细胞,差异有显著性(P<0.05)。结论首次诱导MTX耐药以抑制DHFR基因为主。DHFR基因表达具有手性差异。DHFR基因的检测有望作为肿瘤治疗监测的指标。Objective To study the relationship between methotrexate（MTX） enantiomers resistance and levels of dihydrofolate reductase（DHFR） mRNA.Methods A549 cells were exposed to intermittently and progressively increasing dose of the two enantiomers of MTX.The expression of DHFR gene was assayed by real-time fluorescence quantitative polymerase chain reaction（FQ-PCR）.Results The resistant indexes of cell lines were different for L-（＋）-MTX and D-（-）-MTX enantiomer.D-（-）-MTX resistance cell lines showed higher resistant index than L-（＋）-MTX resistant cell lines.The expression level of DHFR mRNA in the resistant cell lines was less than that of parent cells at the concentration of 15 μmol/L of both L-（＋）-and D-（-）-MTX enantiomer（P0.05）.The expression level of DHFR mRNA in resistant cell lines was relatively high at increasing concentration of 35 μmol/L and 45 μmol/L of D-（-）-MTX.The results of the FQ-PCR revealed that the MTX resistance was associated with increased expression of DHFR mRNA.Conclusion The expression of DHFR gene was inhibited after the cell lines induced by 15 μmol/L of D-（-）-MTX enantoimers in MTX resistant cell line.The expression of DHFR gene showed significant difference in chirality.DHFR mRNA should be examined during MTX treatment,which could be helpful to prognosticate the resistant status of cell line.

关 键 词：二氢叶酸还原酶 氨甲蝶呤 对映体 耐药 实时荧光定量PCR 

分 类 号：R734.2[医药卫生—肿瘤]