CAJ | 학술논문

目的研究不同甲氨蝶呤(MTX)对映体耐药与叶酰聚谷氨酸合成酶(FPGS)基因水平表达的关系.方法用大剂量冲击递增结合低剂量持续诱导法诱导获得两组含不同构型15～55μmol/L浓度的MTX对映体[L-(+)-MTX和D-(-)-MTX]耐药的细胞系,细胞为人源非小细胞性肺癌A549细胞,用四甲基偶氮唑盐(MTT)法检测各细胞系的耐药指数;用实时荧光定量聚合酶链反应(RFQ-PCR)方法检测这两组各细胞系中胞质型FPGS(cFPGS)和线粒体型FPGS(mFPGS)基因的相对含量.结果 D-(-)-MTX耐药细胞组耐药指数高于L-(+)-MTX耐药细胞组(32.7±9.3比11.5±2.9,P＜0.05),L-(+)-MTX/A549细胞系耐药指数均在5～15之间,为中度耐药,而D-(-)-MTX/A549细胞系耐药指数均＞15,为高度耐药.在D-(-)-MTX和L-(+)-MTX两组耐药细胞系中,mFPGS表达水平仪在MTX为15 μmol/L时差异无统计学意义,在MTX其他各浓度点两组间差异均有统计学意义(25 μmol/L:2.3±0.9比1.3±0.7,35 μnol/L:2.6±0.3比1.1±0.9,45 μmol/L:1.4±0.8比1.0±1.0,55 μmol/L;1.0±0.2比0.2±0.1均P＜0.05);cFPGS表达水平在MTX为15μmol/L时两组间差异也同样无统计学意义,在25～55 μmol/L浓度区间内D-(-)-MTX/A549细胞系的cFPGS表达与耐药指数呈现高度负相关(r=-0.95,P＜0.05).结论在A549细胞中MTX对映体初次剂量15 μmol/L冲击法诱导获得的对映体耐药与再次接受更大剂量(≥25 μmol/L)MTX诱导获得耐药的机制不同,D-(-)-MTX/A549耐药细胞系表现为更高的耐药性,提示临床使用MTX时应考虑该药物存在手性对映体问题.
목적 연구불동갑안접령(MTX)대영체내약여협선취곡안산합성매(FPGS)기인수평표체적관계.방법 용대제량충격체증결합저제량지속유도법유도획득량조함불동구형15～55μmol/L농도적MTX대영체[L-(+)-MTX화D-(-)-MTX]내약적세포계,세포위인원비소세포성폐암A549세포,용사갑기우담서염(MTT)법검측각세포계적내약지수;용실시형광정량취합매련반응(RFQ-PCR)방법검측저량조각세포계중포질형FPGS(cFPGS)화선립체형FPGS(mFPGS)기인적상대함량.결과 D-(-)-MTX내약세포조내약지수고우L-(+)-MTX내약세포조(32.7±9.3비11.5±2.9,P＜0.05),L-(+)-MTX/A549세포계내약지수균재5～15지간,위중도내약,이D-(-)-MTX/A549세포계내약지수균＞15,위고도내약.재D-(-)-MTX화L-(+)-MTX량조내약세포계중,mFPGS표체수평의재MTX위15 μmol/L시차이무통계학의의,재MTX기타각농도점량조간차이균유통계학의의(25 μmol/L:2.3±0.9비1.3±0.7,35 μnol/L:2.6±0.3비1.1±0.9,45 μmol/L:1.4±0.8비1.0±1.0,55 μmol/L;1.0±0.2비0.2±0.1균P＜0.05);cFPGS표체수평재MTX위15μmol/L시량조간차이야동양무통계학의의,재25～55 μmol/L농도구간내D-(-)-MTX/A549세포계적cFPGS표체여내약지수정현고도부상관(r=-0.95,P＜0.05).결론 재A549세포중MTX대영체초차제량15 μmol/L충격법유도획득적대영체내약여재차접수경대제량(≥25 μmol/L)MTX유도획득내약적궤제불동,D-(-)-MTX/A549내약세포계표현위경고적내약성,제시림상사용MTX시응고필해약물존재수성대영체문제.
Objective To investigate the relationship between the resistance of methotrexate (MTX) enantiomer and the gene expression levels of folylpolyglutamate synthetase (FPGS).Methods The cell lines of MTX enantiomer resistance from 15 -55 μmol/L were obtained when the A549 cell lines were exposed intermittently and progressively to an incremental dose of each MTX enantiomer.The resistant index of MTX resistance cell lines were detected by MTT.The gene expressions of FPGS in cytoplasm and mitochondria were detected by real-time quantitative polymerase chain reaction (PCR).Results The resistance indice of D-( - )-MTX resistant cell lines were higher than those of L-( + )-MTX resistant cells (32.7±9.3 vs 11.5 ±2.9,P ＜0.05).The resistant indice of L-( + )-MTX /A549 were from 5 to 15,which mean the middle resistance.The resistant indice of D-( - )-MTX/A549 were more than 15,which mean the severe resistance.The expression of mFPGS had difference between resistant cell lines of L-( + )and D-( - )-MTX except at 15 μmol/L MTX (at 25 μmol/L,1.3 ±0.7 vs.2.3 ±0.9;at 35 μmol/L,1.1 ±0.9 vs.2.6±0.3;at 45 μmol/L,1.0±1.0 vs.1.4±0.8;at 55 μmol/L,0.2±0.1 vs.1.0±0.2;all P＜0.05).The expressions of cFPGS had no difference between resistant cell lines of L-( + ) and D-( - )-MTX at 15 μmol/L MTX,while at 25 -55 μmol/L,the cFPGS levels and resistance indice of D-( - )-MTX/A549 resistant cell lines showed a highly negative correlation ( r = - 0.95,P＜0.05 ).Conclusion There may be a different mechanism between the first time treatment with 15 μmol/L dosage and the continual treatment with more than 25 μmol/L dosage in A549 cell lines.There had higher resistant index in D-( - )-MTX/A549 cell line than in L-( + )-MTX/A549 cell line.The results indicated that the difference in chirality should be considered in clinical treatment with MTX.