毛细管电泳免疫—激光诱导荧光检测技术测定氨甲蝶呤对映体耐药A549细胞株中叶酰聚谷氨酸合成酶
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摘要
背景:氨甲蝶呤(Methotrexate,MTX)是许多叶酸途径抗肿瘤药物的前体,存在L-(+)-MTX和D-(-)-MTX 2种形式的对映体,广泛应用于淋巴瘤、急性白血病和肺癌等多种肿瘤的治疗,作为MTX代谢途径中的关键酶之一的细胞内叶酰聚谷氨酸合成酶(folylpolyglutamate synthetase,FPGS),其含量和活性直接与MTX作用效果相关,已成为当今研究的热点,但有关FPGS定量检测的报道甚少。
目的:建立一种FPGS定量检测方法,优化该方法实验条件,对该方法的特异性,分离度及灵敏度等进行方法学评价,并对其检测结果进行统计分析。用该方法检测不同氨甲蝶呤(MTX)对映体作用肺癌A549细胞株前后细胞株内FPGS表达含量,观察FPGS含量变化,为深入探讨肿瘤耐药机制提供新的研究平台。
方法:首先利用大剂量浓度递增冲击法诱导耐药的方法分别诱导建立25μmol/L L-(+)-MTX耐药细胞株和25μmol/L D-(-)-MTX耐药细胞株,以敏感细胞株作为细胞内FPGS表达含量的对照;对湿重均约为100mg的敏感细胞株、25μmol/L L-(+)-MTX耐药细胞株和25μmol/L D-(-)-MTX耐药细胞株的三组细胞株采用液氮中反复冻融的方法,完全破坏细胞,获取细胞蛋白上清提取液;提取液经经过SDS-聚丙烯酰胺电泳(SDS-PAGE)和免疫印迹(Western blot)分析;细胞提取液在室温暗室里与经过透析标记法,采用异硫氰酸荧光素( fluorescein isothiocynate,FITC)衍生标记的FPGS抗体反应30 min,使标记抗体与FPGS发生特异性非竞争性免疫反应;反应后立即采取毛细管胶束电动色谱(micellar electrokinetic capillary chromatography,MECC )法结合激光诱导荧光(laser-induced fluorescence,LIF)检测技术的毛细管电泳免疫-激光诱导荧光检测技术(immunity analysis of capillary electrophoresis-laser-induced fluorescence,CEIA-LIF)分离检测FITC衍生标记FPGS以及FPGS抗原抗体复合物,检测MTX不同对映体作用肺癌A549细胞株中FPGS表达含量的。
结果:经SDS-PAGE确认FPGS分子量约为66 000D,WB实验确认FPGS与其特异性抗体没有其他条带出现,并通过凝胶成像系统初步定量:25μmol/L L-(+)-MTX和25μmol/L D-(-)-MTX诱导耐药细胞中FPGS的表达含量分别为敏感细胞的52.14%和60.06%;CEIA-LIF检测方法分离标记FPGS抗体与免疫复合物的时间分别为7.1min、8.9min,且在10min内即完成蛋白的分离和检测,分离度R=4.5;CEIA-LIF检测FPGS的方法在敏感细胞株内检出限为0.6788 mg/μl细胞;运用CEIA-LIF检测细胞内FPGS的表达含量的方法,计算得出25μmol/L L-(+)-MTX和25μmol/L D-(-)-MTX诱导耐药细胞中FPGS的表达含量分别为敏感细胞的46.59%和48.36%,该结果和经典的WB初步定量结果,经SPSS 13.0 Wald模式处理软件分析得知P>0.05,说明两者没有显著差异。
结论:CEIA-LIF检测FPGS方法具有检测速度快,测定时间短的特点。且通过经典的WB法做对照,该方法具有高度的特异性和FPGS定量的准确性。通过该方法中运用了FITC标记技术、MECC分离技术以及LIF检测系统,使检测特异性、敏感度大大的提高。通过建立的CEIA-LIF检测MTX不同对映体作用肺癌A549细胞株中FPGS表达含量,得出L-(+)-MTX和D-(-)-MTX诱导耐药后细胞株中FPGS表达含量较敏感细胞株明显减少,说明MTX耐药细胞株FPGS表达含量受损。
Background: Methotrexate (MTX) is a significant drug precursors of folic acid-like anticancer, with two kinds of enantiomers: L-(+)-MTX and D-(-)-MTX,which was widely used in treatment of lymphoma, acute leukemia and lung cancer and other tumor.As one of the key-enzymes in folic acid metabolic pathway, Folylpolyglutamate synthetase (FPGS) content and activity directly associated with the effects of MTX. At present, many researches concentrate themselves on FPGS study, but the quantitative detection of FPGS was rarely reported.
Objective: Firstly, to design a detection method of FPGS, secondly optimize the experiment conditions, thirdly evaluate the specificity, separation, and sensitivity of the designed method,lastly to analyse the test results by statistical software. Observing the change of FPGS contents in the drug-resistant cell strains that evolved from A549 after being induced by different methotrexate (MTX) enantiomer with this method, to provide new research platform for future study of tumor drug resistance mechanisms
Methods: First, 25μmol / L L-(+)-MTX drug resistant cell strains and 25μmol / L D-(-)-MTX drug resistant cell strains were established by using large doses of incremental drug method, and sensitive cell strains as a control in FPGS expression. All the three cell strains were repeatedly freezed and thawed by using liquid Nitrogen, completely destroy cells, collect cell protein extract of the supernatantby the way the three strains wet weight about 100 mg,;Analysis the extract with the SDS-polyacrylamide electrophoresis(SDS-PAGE) and Western blot(WB);Use fluorescein isothiocyanate (fluorescein isothiocynate, FITC) derivatives marked FPGS antibody with dialysis method,then reaction with the extract in room temperature and dark room,for 30 min .The non-competitiveimmune reaction have strong specific;after reaction,using immune capillary electrophoresis - laser-induced fluorescence detection technology (CEIA-LIF) isolate marked FPGS antibody and FPGS antigen-antibody mixture, CEIA-LIF ,include capillary micellar electrokinetic chromatography (MEKC) detection technology and laser-induced fluorescence detector (LIF) technology, can detect the FPGS expression in different MTX enantiomers affects the lung cancer A549 cell strains.
Results: FPGS molecular weight is about 66 000D with confirmation by SDS-PAGE experiments ,and there was no other band came forth which was confirmed by WB experiments. After the gel imaging system preliminary quantitive analysis, FPGS expressions of 25 mol / L-(+)-MTX drug resistant cell strains and 25 mol / L D-(-)-MTX drug resistant cell strains are 52.14%and 60.06% compare with sensitive cell strains respectively.Useing CEIA-LIF test method, the process of separation takes 7.1 min in separation of marked FPGS antibody, at the same time it takes 8.9 min in separation of immune complexes. In both process it can be separated and detected in no more than 10 mins,and the resolution threshold (R) is 4.5.With the method of CEIA-LIF detection cells FPGS expression, the FPGS expressions of 25 mol / L-(+)-MTX drug resistant cell strains and of 25 mol / L D-(-)-MTX drug resistant cell strains are 46.59%and 48.36%respectively,compareed with sensitive cell strains. These results accompanied with the quantitative results of classic WB preliminary were statistical analysesed by the statistical software SPSS (Version:13.0) Weld pattern,P>0.05, demonstrated there no significant difference.
Conclusions: CELA-LIF is of generally fast efficient in detecting FPGS, the determination time is short,and similar to WB method, with high specificity and high accuracy;With fluorescent markered, MECC separation technology and LIF detection system useing in this new method, the detection specificity, sensitivity greatly improved. The expression consents of FPGS in L-(+)-MTX and D-(-)-MTX drug resistant cell strains is damaged and decreased obviously.
目的:建立一种FPGS定量检测方法,优化该方法实验条件,对该方法的特异性,分离度及灵敏度等进行方法学评价,并对其检测结果进行统计分析。用该方法检测不同氨甲蝶呤(MTX)对映体作用肺癌A549细胞株前后细胞株内FPGS表达含量,观察FPGS含量变化,为深入探讨肿瘤耐药机制提供新的研究平台。
方法:首先利用大剂量浓度递增冲击法诱导耐药的方法分别诱导建立25μmol/L L-(+)-MTX耐药细胞株和25μmol/L D-(-)-MTX耐药细胞株,以敏感细胞株作为细胞内FPGS表达含量的对照;对湿重均约为100mg的敏感细胞株、25μmol/L L-(+)-MTX耐药细胞株和25μmol/L D-(-)-MTX耐药细胞株的三组细胞株采用液氮中反复冻融的方法,完全破坏细胞,获取细胞蛋白上清提取液;提取液经经过SDS-聚丙烯酰胺电泳(SDS-PAGE)和免疫印迹(Western blot)分析;细胞提取液在室温暗室里与经过透析标记法,采用异硫氰酸荧光素( fluorescein isothiocynate,FITC)衍生标记的FPGS抗体反应30 min,使标记抗体与FPGS发生特异性非竞争性免疫反应;反应后立即采取毛细管胶束电动色谱(micellar electrokinetic capillary chromatography,MECC )法结合激光诱导荧光(laser-induced fluorescence,LIF)检测技术的毛细管电泳免疫-激光诱导荧光检测技术(immunity analysis of capillary electrophoresis-laser-induced fluorescence,CEIA-LIF)分离检测FITC衍生标记FPGS以及FPGS抗原抗体复合物,检测MTX不同对映体作用肺癌A549细胞株中FPGS表达含量的。
结果:经SDS-PAGE确认FPGS分子量约为66 000D,WB实验确认FPGS与其特异性抗体没有其他条带出现,并通过凝胶成像系统初步定量:25μmol/L L-(+)-MTX和25μmol/L D-(-)-MTX诱导耐药细胞中FPGS的表达含量分别为敏感细胞的52.14%和60.06%;CEIA-LIF检测方法分离标记FPGS抗体与免疫复合物的时间分别为7.1min、8.9min,且在10min内即完成蛋白的分离和检测,分离度R=4.5;CEIA-LIF检测FPGS的方法在敏感细胞株内检出限为0.6788 mg/μl细胞;运用CEIA-LIF检测细胞内FPGS的表达含量的方法,计算得出25μmol/L L-(+)-MTX和25μmol/L D-(-)-MTX诱导耐药细胞中FPGS的表达含量分别为敏感细胞的46.59%和48.36%,该结果和经典的WB初步定量结果,经SPSS 13.0 Wald模式处理软件分析得知P>0.05,说明两者没有显著差异。
结论:CEIA-LIF检测FPGS方法具有检测速度快,测定时间短的特点。且通过经典的WB法做对照,该方法具有高度的特异性和FPGS定量的准确性。通过该方法中运用了FITC标记技术、MECC分离技术以及LIF检测系统,使检测特异性、敏感度大大的提高。通过建立的CEIA-LIF检测MTX不同对映体作用肺癌A549细胞株中FPGS表达含量,得出L-(+)-MTX和D-(-)-MTX诱导耐药后细胞株中FPGS表达含量较敏感细胞株明显减少,说明MTX耐药细胞株FPGS表达含量受损。
Background: Methotrexate (MTX) is a significant drug precursors of folic acid-like anticancer, with two kinds of enantiomers: L-(+)-MTX and D-(-)-MTX,which was widely used in treatment of lymphoma, acute leukemia and lung cancer and other tumor.As one of the key-enzymes in folic acid metabolic pathway, Folylpolyglutamate synthetase (FPGS) content and activity directly associated with the effects of MTX. At present, many researches concentrate themselves on FPGS study, but the quantitative detection of FPGS was rarely reported.
Objective: Firstly, to design a detection method of FPGS, secondly optimize the experiment conditions, thirdly evaluate the specificity, separation, and sensitivity of the designed method,lastly to analyse the test results by statistical software. Observing the change of FPGS contents in the drug-resistant cell strains that evolved from A549 after being induced by different methotrexate (MTX) enantiomer with this method, to provide new research platform for future study of tumor drug resistance mechanisms
Methods: First, 25μmol / L L-(+)-MTX drug resistant cell strains and 25μmol / L D-(-)-MTX drug resistant cell strains were established by using large doses of incremental drug method, and sensitive cell strains as a control in FPGS expression. All the three cell strains were repeatedly freezed and thawed by using liquid Nitrogen, completely destroy cells, collect cell protein extract of the supernatantby the way the three strains wet weight about 100 mg,;Analysis the extract with the SDS-polyacrylamide electrophoresis(SDS-PAGE) and Western blot(WB);Use fluorescein isothiocyanate (fluorescein isothiocynate, FITC) derivatives marked FPGS antibody with dialysis method,then reaction with the extract in room temperature and dark room,for 30 min .The non-competitiveimmune reaction have strong specific;after reaction,using immune capillary electrophoresis - laser-induced fluorescence detection technology (CEIA-LIF) isolate marked FPGS antibody and FPGS antigen-antibody mixture, CEIA-LIF ,include capillary micellar electrokinetic chromatography (MEKC) detection technology and laser-induced fluorescence detector (LIF) technology, can detect the FPGS expression in different MTX enantiomers affects the lung cancer A549 cell strains.
Results: FPGS molecular weight is about 66 000D with confirmation by SDS-PAGE experiments ,and there was no other band came forth which was confirmed by WB experiments. After the gel imaging system preliminary quantitive analysis, FPGS expressions of 25 mol / L-(+)-MTX drug resistant cell strains and 25 mol / L D-(-)-MTX drug resistant cell strains are 52.14%and 60.06% compare with sensitive cell strains respectively.Useing CEIA-LIF test method, the process of separation takes 7.1 min in separation of marked FPGS antibody, at the same time it takes 8.9 min in separation of immune complexes. In both process it can be separated and detected in no more than 10 mins,and the resolution threshold (R) is 4.5.With the method of CEIA-LIF detection cells FPGS expression, the FPGS expressions of 25 mol / L-(+)-MTX drug resistant cell strains and of 25 mol / L D-(-)-MTX drug resistant cell strains are 46.59%and 48.36%respectively,compareed with sensitive cell strains. These results accompanied with the quantitative results of classic WB preliminary were statistical analysesed by the statistical software SPSS (Version:13.0) Weld pattern,P>0.05, demonstrated there no significant difference.
Conclusions: CELA-LIF is of generally fast efficient in detecting FPGS, the determination time is short,and similar to WB method, with high specificity and high accuracy;With fluorescent markered, MECC separation technology and LIF detection system useing in this new method, the detection specificity, sensitivity greatly improved. The expression consents of FPGS in L-(+)-MTX and D-(-)-MTX drug resistant cell strains is damaged and decreased obviously.
引文
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3. Evans WE, et al. Conventional compared with individualized chemotherapy for childhood acute lymphoblastic leukemia. New England J Medicine. 1998; 338:499-505.
4. Ackland SP, et al. High-dose methotrexate: a critical reappraisal. J Clin Oncol. 1987; 5:2017–2031.
5. Evans WE, et al. Clinical pharmacodynamics of high-dose methotrexate in acute lymphoblastic leukemia: identification of a relation between concentration and effect. New England J Medicine. 1986; 314:471-477.
6. Niemeyer CM, et al. Low-dose versus high-dose methotrexate during remission induction in childhood acute lymphoblastic leukemia (Protocol 81-01 update). Blood. 1991; 78:2514-2519.
7.陈静,等.急性白血病氨甲蝶呤耐药及化疗个体化初探.中华血液学杂志.. 2002; 23(1): 35-36.
8. Barry M. Trost, Asymmetric catalysis: an enabling science. PNAS. 2004;101(15):5348-5355.
9. Cramer SM, et al. Occurrence and significance of D-methotrexate as a contaminant of commercial methotrexate. Cancer Res. 1984; 44(5):1843-1846.
10. Cramer SM, et al. Occurrence and significance of D-methotrexate as a contaminant of commercial methotrexate. Cancer Res. 1984; 44(5):1843-1846.
11.沈佐君,等.高效毛细管电泳法在血清氨甲蝶呤浓度检测中的应用.中华检验医学杂志. 2005; 28(2):206-208.
12.何晓东,等.毛细管区带电泳法拆分氨甲蝶呤手性对映体.检验医学与临床. 2005;2 (1):3-5.
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2. Schilsky RL, et al. Methotrexate: an effective agent for treating cancer and building careers. The polyglutamate era. Stem Cells. 1996; 14(1): 29-32.
3. Evans WE, et al. Conventional compared with individualized chemotherapy for childhood acute lymphoblastic leukemia. New England J Medicine. 1998; 338:499-505.
4. Ackland SP, et al. High-dose methotrexate: a critical reappraisal. J Clin Oncol. 1987; 5:2017–2031.
5. Evans WE, et al. Clinical pharmacodynamics of high-dose methotrexate in acute lymphoblastic leukemia: identification of a relation between concentration and effect. New England J Medicine. 1986; 314:471-477.
6. Niemeyer CM, et al. Low-dose versus high-dose methotrexate during remission induction in childhood acute lymphoblastic leukemia (Protocol 81-01 update). Blood. 1991; 78:2514-2519.
7.陈静,等.急性白血病氨甲蝶呤耐药及化疗个体化初探.中华血液学杂志.. 2002; 23(1): 35-36.
8. Barry M. Trost, Asymmetric catalysis: an enabling science. PNAS. 2004;101(15):5348-5355.
9. Cramer SM, et al. Occurrence and significance of D-methotrexate as a contaminant of commercial methotrexate. Cancer Res. 1984; 44(5):1843-1846.
10. Cramer SM, et al. Occurrence and significance of D-methotrexate as a contaminant of commercial methotrexate. Cancer Res. 1984; 44(5):1843-1846.
11.沈佐君,等.高效毛细管电泳法在血清氨甲蝶呤浓度检测中的应用.中华检验医学杂志. 2005; 28(2):206-208.
12.何晓东,等.毛细管区带电泳法拆分氨甲蝶呤手性对映体.检验医学与临床. 2005;2 (1):3-5.
13. Schultz MN , Kennedy RT. A nal Chem , 1993 ,65∶3161-3165.
14. Chen FTA,Evangelista RA. Clin Chem , 1994 ,40∶1819.
15. Shimura K,KargerBL. Anal Chem , 1994 ,66∶9
16. Bruce A, Joseph D. Density of surface immunoglobulin and capping on rat B lymphocytes. J Exp ,Med,1979,149:416-23.
17.张津,施维,王磊,等.毛细管区带电泳峰形变化规律的研究.色谱,1997,15(3):197-200.
18. McGuire JJ, Russell CA, Balinsha M. Human cytosolic and mitochondrial folypolyglutamate synthetase are electrophoretically distinct. J Biological Chemistry, 2000,275:13012-16.
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