【作者】 孙利；

【导师】 沈佐君；

【作者基本信息】 安徽医科大学 ， 临床检验诊断学， 2011， 硕士

【摘要】 目的:探讨叶酰聚谷氨酸合成酶(FPGS)在氨甲蝶呤(MTX)对映体(L-(+)-MTX和D-(-)–MTX)耐药A549细胞株中的表达,并应用于临床病例。方法:用SYBR Green I为荧光染料,以β-actin作参照,建立检测FPGS mRNA的实时荧光定量PCR方法;根据循环阈值(Ct值)、标准曲线相关系数、斜率、重复性曲线、熔解曲线、扩增效率曲线等进行方法学评价;并用该方法检测MTX对映体耐药细胞株及应用MTX耐药的白血病骨髓细胞中FPGS基因表达;用蛋白免疫印迹方法测定肺癌A549细胞株和MTX对映体耐药A549细胞株中的FPGS蛋白表达。结果:建立的标准曲线Ct值与模板浓度有良好的线性关系,FPGS和β-actin标准曲线相关系数分别为0.9968和0.9987,斜率分别为-3.595和-3.740,批内变异系数(CV)为1.27%～2.95%,批间CV为3.82%,熔解曲线均呈单个特异峰,扩增效率相似(斜率为0.0217);15μmol/L、35μmol/L、55μmol/L各浓度点L-(+)-MTX和D-(-)–MTX两组间FPGS mRNA表达水平差异均有统计学意义(0.80±0.09 vs 2.04±0.34,3.43±0.44 vs 8.11±0.87,2.00±0.22 vs 0.59±0.11, P<0.05);15μmol/L L-(+)-MTX和D-(-)–MTX两组间FPGS蛋白表达水平差异有统计学意义(0.85±0.12 vs 1.62±0.24,P<0.05);浓度为35μmol/L、55μmol/L时, L-(+)-MTX和D-(-)–MTX两组间蛋白表达水平差异均无统计学意义(P>0.05);白血病患者应用MTX耐药后FPGS相对含量是用前的0.35±0.04倍,两组间差异具有统计学意义(P<0.05)。结论:建立的实时荧光定量PCR检测FPGS mRNA的方法重复性好、特异度高、稳定性好、简单易行且成本低廉,可用于FPGS基因含量分析;白血病患者应用MTX耐药前后骨髓细胞中FPGS基因表达发生了变化;MTX诱导耐药后细胞株中FPGS在mRNA和蛋白水平上均发生了变化且在两种对映体细胞株间具有手性差异。更多还原

【Abstract】 Objective:To study the expression of folylpolyglutamate synthetase (FPGS) in methotrexate enantiomers resistant A549 cell lines (L-(+)-MTX和D-(-)–MTX)and the application of clinical specimens.Methods:A real-time fluorescence quantitative PCR method for detection of FPGS mRNA was established using SYBR Green I as fluorescence andβ-actin as reference. The method was evaluated by cycle threshold (Ct), correlation coefficient, slope, repeatability curve, melting curve and amplification efficiency curve. The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method. The expression of FPGS on protein level was determined by Western-Blot in lung cancer A549 cells, and methotrexate enantiomers resistant A549 cells.Results:The standard curves indicated the high linear relationship between cycle threshold and template concentration was observed. The correlation coefficients of FPGS andβ-actin were 0.9968 and 0.9987, and the slopes were -3.595 and -3.740, respectively. The inter-coefficient of variation was from 1.27% to 2.95%. The intra-coefficient of variation was 3.82%. The method was characterized of specific melting curve and similar amplification efficiency (slope =0.0217). The expression level of FPGS mRNA was statistical different in various MTX concentration resistant cell lines (0.80±0.09 vs 2.04±0.34,3.43±0.44 vs 8.11±0.87,2.00±0.22 vs 0.59±0.11, P<0.05)。The protein expression level of FPGS was 0.85±0.12 and 1.62±0.24 folds in L-(+)-MTX/A549 cells and D-(-)-MTX/A549 cells with the concentration of drug resistance is 15μmol/L ,compared with lung cancer A549 cells.Statistical difference was observed between two groups (P<0.05). However there had no statistical difference in MTX enantiomers resistant cell line with the concentration of drug resistance are 35μmol/L and 55μmol/L (P>0.05). After treated with methotrexate(group B), the expression level of FPGS mRNA was 0.35±0.04 folds compared with those before treatment (group A) among methotrexate resistant leukemia patients. Statistical difference was observed between group A and group B(P<0.05).Conclusion:The real-time fluorescence quantitative PCR has many advantages, such as a reproductive, specific, stable, simple and cheap method, which is suitable for the quantification of FPGS. The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow have been changed. The expression level of FPGS in drug resistant cells have been changed, especially significant difference in enantiomer-resistant cells are appeared.更多还原