实时荧光定量PCR法检测MTX对映体耐药A549细胞株及白血病患者骨髓细胞中FPGSmRNA的表达  

Development of a real-time fluorescence quantitative PCR method for detection of FPGS mRNA expression in methotrexate enantiomer-resistant A549 cell lines and patients with leukemia

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

机构地区：[1]安徽医科大学附属省立医院安徽省临床检验中心,合肥230001

出　　处：《中华检验医学杂志》2011年第8期722-726,共5页Chinese Journal of Laboratory Medicine

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（20052040）

摘　　要：目的建立实时荧光定量PCR检测肿瘤细胞中FPGSmRNA表达的方法，研究MTX对映体[L-（＋）-MTX和D-（-）-MTX]耐药细胞株中FPGS的基因表达差异，并用于观察白血病患者MTX治疗耐药前后FPGSmRNA表达水平的变化。方法用SYBRGreenI为荧光染料，以β—actin作参照，建立检测FPGSmRNA的实时荧光定量PCR方法。根据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14例白血病患者骨髓细胞中FPGSmRNA的表达。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）；L．（＋）-MTX耐药细胞和D．（-）-MTX耐药细胞中FPGSmRNA相对含量分别为（3．51±0．66）和（0．16±0．01），A549亲本细胞（S）中FPGSmRNA相对含量为（1．00±0．31），差异有统计学意义（F=64．45，P〈0．01）；L-（＋）-MTX耐药细胞和D．（-）-MTX耐药细胞间FPGSmRNA相对含量差异有统计学意义（q=9．29，P〈0．01）。白血病患者应用MTX治疗耐药后，FPGSmRNA表达水平为（0．35±0．04），用药前为（1．00±0．44），差异有统计学意义（t=8．83，P〈0．01）。结论建立的实时荧光定量PCR检测FPGSmRNA的方法重复性好、特异度高，可用于FPGSmRNA含量分析。MTX诱导耐药后细胞株及白血病患者骨髓细胞中FPGSmRNA表达发生了变化，MTX2种对映体形式在细胞耐药机制中可能发挥了不同的作用。Objective To establish a real-time fluorescence quantitative PCR method for detection of the different expression level of FPGS in methotrexate enantiomer-resistant A549 cell lines, and observe FPGS mRNA expression in patients with leukemia. Methods A real-time fluorescence quantitative PCR method for detection FPGS mRNA was established using SYBR Green I as fluorescence and 13-actin as reference. The method was evaluated by 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. Results The standard curves had a high linear relationship between cycle threshold and template concentration. The correlation coefficients of FPGS and 13-actin were 0. 996 8 and 0. 998 7, 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 with specific melting curve and similar amplification efficiency （slope was 0. 021 7 ）. The relative contents of FPGS mRNA were （3.51± 0. 66）, （0. 16 ±0. 01 ） and （ 1.00±0. 31 ） in L-（ ＋ ）-MTX/A549 cells（L）, D-（-）-MTX/A549 cells（D） and A549 parent cells, and there was statistically difference among the three groups（ F = 64. 45 ,P 〈 0. 01 ）. Statistical difference was observed between L and D （ q = 9. 29, P 〈 0. 01 ）. After treated with MTX, the expression level of FPGS mRNA was （0. 35±0. 04 ） in methotrexate resistant leukemia patients, compared with （ 1.00 ± 0. 44） before treatment. Statistical difference was observed （ t = 8.83,P 〈 0. 01 ）. Conclusions The real-time fluorescence quantitative PCR is suitable for the quantification of FPGS. The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow and drug resistant cells are different. Two enantiomer

关 键 词：细胞系 肿瘤 白血病 甲氨蝶呤 肽合酶类 抗药性 肿瘤 聚合酶链反应 立体异构现象 

分 类 号：R733.7[医药卫生—肿瘤]